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Michigan State
University
This is to certify that the
thesis entitled

EFFECTS OF TIMING OF ARTIFICIAL INSEMINATION AND
SITE OF SEMEN DEPOSITION ON FERTILITY IN
LACTATING DAIRY COWS

presented by

MELISSA WOLF MACFARLANE

has been accepted towards fulﬁllment
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MS. degree in Animal Science

 

 

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6/01 c-JCIRCIDaIeDuest-p. 1 5

EFFECTS OF TIMING OF ARTIFICIAL INSEMINATION AND SITE OF
SEMEN DEPOSITION ON FERTILITY IN LACTATING DAIRY COWS
AND GENDER RATIO OF RESULTING OFFSPRING

By

Melissa Wolf Macfarlanc

A THESIS

Submitted to
Michigan State University
in partial ﬁJlﬁllment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

2003

ABSTRACT

EFFECTS OF TIMING OF ARTIFICIAL INSEMINATION AND SITE OF SEMEN
DEPOSITION ON FERTILITY IN LACTATIN G DAIRY COWS AND GENDER
RATIO OF RESULTING OFFSPRING

By

Melissa Wolf Macfarlane

This study was designed to determine the effect of prolonged subsistence of
sperm in the female reproductive tract prior to ovulation in lactating dairy cows, with two
different sites of AI, on pregnancy rates per AI (PR/AI) and resulting gender ratio. The
Ovsynch program was utilized for precise timing of AI in relation to ovulation (24-32 h
following ﬁnal GnRH). Cows from three commercial dairies (n=1603) were assigned to
one of four treatment groups (2 x 2) based on parity and days in milk (DIM). Cows were
inseminated in the uterine body (B) or uterine horn (H) 8 h before (B or H - 8 h groups),
or 16 h aﬁer (B or H 16 h groups), the ﬁnal GnRH injection of Ovsynch. Ultrasound was
used to diagnose pregnancy at 28 and 56 d after AI. There was no effect of site of Al on
PR/AI, so B and H groups were combined within -8 and 16 groups. More female calves
than male calves (65: 35 female: male ratio) were born in the -8 h group (P < 0.01).
However, the 16 h group had a greater (P < 0.01) PR/AI (39 % vs. 29 %) than the -8 h
group. To evaluate bull fertility at farm 3, service sires were selected based on Estimated
Relative Conception Rate. There was an effect of service sire at -8 and 16 h on PR/AI
and embryonic survival from 28 to 56 d (P < 0.01). In summary, prolonged subsistence
of sperm in the female tract diminishes fertility and procreates more female offspring.

Some service sires attenuated this diminished fertility and improved embryonic survival.

I dedicate this thesis to Bruce J. Macfarlane, my best friend
and partner for life. Together we can overcome

any challenge in life, love and research.

iii

ACKNOWLEDGEMENT

I would like to thank my major professor Dr. Richard Pursley. His knowledge,
patience and open door policy have contributed to my knowledge of the dairy industry,
reproductive physiology and my budding research savvy. Also my appreciation goes out
to my guidance committee, Dr. George Smith, Dr. Guilherme Rosa, Dr. Jeanne Burton
and Dr. Kent Ames. Additionally, I would like to acknowledge Dr. Rosa for his help
with the statistical analysis and Dr. Allen Tucker for his help with editing.

Funding for this research was provided by an Initiative for Future Agriculture and
Food Systems (IFAF S) grant. Additionally, Merial Ltd. and Pharmacia-Upjohn Co.
donated the Cystorelin® and Lutalyse® used in the synchronization protocol. I would
also like to thank Select Sires, ABS and Genex for donating semen. I thank Dr. Karen
Chou and Melissa Vadnais for the use of their lab and assistance with post-thaw semen
analysis.

I would like to thank the three dairies that participated in this study: Nobis,
Cimarron and Verba—Hoff. I appreciate the help of the managers and employees who
worked closely with us to accomplish our research objectives. I would also like to thank
Gabe Papoi an MSU undergraduate, who spent long hours with us collecting data at
Vreba-Hoff Dairy.

My husband, 8.]. Macfarlane, had an integral part of the collection of data for this
study. Through long days and thousands of inseminations, we collected an enormous

amount of data, lived in Kansas and managed to stay married. I am so grateful for his

iv

continued love and support. He has made many sacriﬁces in order for me to achieve my
Master of Animal Science Degree. Thank you.

I want to thank my parents, Chet and Terrie Wolf who taught me good work ethic
and for always encouraging me to challenge myself. Thank you to my big brothers
Randy and Brian for instilling resilience.

Finally, I want to thank our families and friends back on the west coast. They
have been very supportive, yet maintained a high level of encouragement for rapid
progress. Also, to all our new found friends and family in Michigan thank you so much
for making our stay here in Michigan one that we will never forget and will always

cherish.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES ............................................................................................................ x

LIST OF ABBREVIATIONS ............................................................................................ xi

INTRODUCTION .............................................................................................................. 1
CHAPTER 1

REVIEW OF LITERATURE ............................................................................................. 4

Part I: Timing of Artiﬁcial Insemination (AI) ............................................................... 5

Optimal Time of AI in Relation to Estrus or Ovulation .............................. 5

Importance of Increasing Female Oﬂspring ............................................... 6

Sperm Survival in the Female Reproductive Tract ..................................... 8

Semen Quality and Quantity ....................................................................... 8

Sperm Capacitation and Binding .............................................................. 10

Summary ................................................................................................... 12

Part II Site of Semen Deposition .................................................................................. 12

Sperm Transport in the Female ................................................................ 15

Uterine Environment and Fertility ............................................................ l6

Pitfalls of Uterine Horn Insemination .......................................... 17

Summary ................................................................................................... 18

Part III Physiological and Management Factors That May Affect Fertility ................ l8

Genetics ..................................................................................................... 19

Stress ......................................................................................................... l9

Parity ......................................................................................................... 20

Retrograde Sperm Loss ............................................................................. 21

Phagocytosis ............................................................................................. 22

Locomotion Scores .................................................................................... 22

Body Condition Scores and Nutrition ....................................................... 23

Bovine Somatotropin ................................................................................. 25

Retained Placenta and Dystocia ............................................................... 26

Twinning ................................................................................................... 27

Milk Production, Days in Milk, and Milking Frequency .......................... 28

Summary ................................................................................................... 29

vi

CHAPTER 2

MATERIALS AND METHODS ...................................................................................... 31
Statistical Analysis .................................................................................... 37
RESULTS ......................................................................................................................... 40
DISCUSSION ................................................................................................................... 60
Effect of Extending Sperm Subsistence in the Uterus / oviduct prior to
ovulation ................................................................................................... 60
Effect of Site of Semen Deposition on Fertility ......................................... 64
Effect of Other Physiological and Management Factors on Fertility ...... 66
CONCLUSION ................................................................................................................. 70
BIOGRAPHY ................................................................................................................... 73
REFERENCES ................................................................................................................. 74

vii

LIST OF TABLES

Table 1. Sample size for each of the four treatment groups ............................................. 32

Table 2. The effect of farm on percent of lactating dairy cows pregnant at 28 and 5 6 (1
following AI ...................................................................................................................... 43

Table 3. Effect of timing of AI relative to the ﬁnal GnRH injection of Ovsynch and site
of insemination on percent lactating dairy cows pregnant at 28 and 56 d ........................ 44

Table 4. Percent female and male offspring born to lactating dairy cows at Farm 1 and 2,
inseminated at -8 h or 16 h relative to the ﬁnal GnRH injection of Ovsynch .................. 45

Table 5. Effects of service sire and timing of AI relative to ﬁnal GnRH injection of
Ovsynch on percent lactating dairy cows pregnant at 28 and 56 (1 following AI at Farm 3
.. ........................................................................................................................................ 46

Table 6. Effects of service sire and site of semen deposition, either uterine body or deep
uterine horn, on percent lactating dairy cows pregnant at 28 and 56 (1 following A1 at

Farm3 .. ........................................................................................................................... 47

Table 7. Semen evaluation (for concentration and motility) for each of the ﬁve bulls used
to AI cows at Farm 3 ......................................................................................................... 48

Table 8. Effect of milk production (above or below the mean) for lst service dairy cows
60-90 (I in lactation on percent pregnant at 28 and 56 (1 following AI. ............................ 49

Table 9. Effect of parity on percent of lactating dairy cows pregnant at 28 and 56 (1
following AI ...................................................................................................................... 50

Table 10. Effect of follicle size at time of insemination on percent single ovulating
lactating dairy cows pregnant at 28 and 56 (1 following AI .............................................. 51

Table 11. Effect of multiple ovulations on percent lactating dairy cows pregnant at 28
and 56 (1 following AI ....................................................................................................... 52

Table 12. Effect of corpus luteum (CL) size at time of insemination on percent lactating
dairy cows pregnant at 28 and 56 d following AI ............................................................. 53

Table 13. Percent lactating dairy cows pregnant at 28 and 56 (1 following AI by site of
insemination and scored for locomotion ........................................................................... 54

viii

Table 14. Effect of number of services on percent lactating dairy cows pregnant at 28 and
56 (1 following AI .............................................................................................................. 55

Table 15. Effect of somatic cell score (SCS) near the time of insemination on percent
lactating dairy cows pregnant at 28 and 56 (1 following AI .............................................. 56

Table 16. Effect of previous calving difﬁculty score on percent lactating dairy cows
pregnant at 28 and 56 d following AI .............................................................................. 57

Table 17. Effect of days in milk (DIM) on percent lactating dairy cows pregnant at 28
and 56 d following AI ....................................................................................................... 58

Table 18. Effect of body condition score (BCS) on percent lactating dairy cows pregnant
at 28 and 56 d following AI .............................................................................................. 59

ix

LIST OF FIGURES

Figure l. The timing and purpose of hormone injections in the Ovsynch program, adapted
from Pursley et al., 1995 ..................................................................................................... 6

Figure 2. The timing for each injection used in the Ovsynch protocol and timing of A1 for
all cows on study ............................................................................................................... 33

AI

am

BCS

bST

CL

CR

DHIA

DIM

DRMS

ERCR

FSH

GnRH

IVF

LH

pm

PR

PR/AI

SCC

SCS

ZP

LIST OF ABBREVIATIONS

artificial insemination

morning

body condition score

bovine somatotropin

corpus luteum

conception rate

day

dairy herd improvement association
days in milk

dairy records management system
estimated relative conception rate
follicle stimulating hormone
gonadotropin releasing hormone
hour

in vitro fertilization

luteinizing hormone

afternoon

pregnancy rate

pregnancy rate per AI

retained placenta

somatic cell count

somatic cell score

zona proteins

xi

INTRODUCTION

A decrease in conception rates (CR) and reproductive efﬁciency has occurred in
the dairy industry over the past 30 years. In the 19705, CR from artiﬁcial insemination
(AI) in multiparous lactating dairy cows was 66% (Spalding et al., 1974). The CR in
multiparous lactating cows decreased to 40 % in the 19803 (Butler and Smith, 1989). In
the last 10 years CR in lactating cows declined to approximately 37 % (Lopez-Gatius,
2003).

Decreased reproductive efﬁciency due to decreased CR reduce dairy farm
proﬁtability by increasing the days open and increases culling of potentially highly
proﬁtable cows (Gwazdauskas etal., 1981; Pecsok et al., 1994; Schindler, 1991).
Decrease in CR also reduces the number of genetically superior replacement heifers born.
To maintain herd size the number of cows culled from a herd must equal the number of
replacement heifers needed. This becomes a challenge if numbers of replacement heifers
produced do not equal numbers of cows leaving the herd.

The current average female: male gender ratio for calves born to multiparous
dairy cows is 46: 54 (Ryan and Boland, 1991). The opportunity to increase the number
of heifer calves born would allow dairy producers to raise more replacements and
potentially increase sustainability and proﬁtability. Pursley et a1. (1998), and
Vasconcelos et a1. (1997) altered the gender ratio in favor of females‘when insemination
occurred approximately 28 h prior to ovulation in lactating dairy cows. Unfortunately,

about a 20% reduction in CR was found for cows inseminated at 28 h compared with AI

at approximately 12 h prior to ovulation. The reduced CR may be due to sperm residing
in the uterus for 28 h prior to fertilization. The extended time in the uterus may challenge
sperm survivability and reduce fertilization capability.

We speculate that producers would not use the 28 h protocol because of the
reduced CR. Therefore, we chose to investigate site of semen deposition as a possible
technique to increase CR. This is particularly important in the treatment groups where
sperm presence in the female reproductive tract is extended. Deposition of semen within
the uterine horn may reduce retrograde sperm loss and possibly decrease exposure of
sperm to phagocytosis in the cervix and uterine body (Mattner, 1968). Senger et al.
(1988) demonstrated a dramatic increase in CR from 45 to 70 % using deep uterine horn
insemination. More recently, Pursley and co-workers (2003; unpublished) increased CR
in lactating dairy cows from 35 to 50 % with deep uterine horn AI. In addition, Diskin
and Pursley (2003; unpublished) increased CR in lactating dairy cows in Ireland from 45
to 50 % with deep uterine horn A1.

The objective of my research was to determine whether timing of AI and deep
uterine horn insemination can be combined to produce an optimal program for dairy
producers to obtain high CR and increase number of heifer calves born.

Additionally, my experiments assessed physiological and management factors
associated with milk production, parity, somatic cell, previous calving information, body

condition and lameness and their effect on fertility in multiparous lactating dairy cows.

The hypotheses for this thesis were:
1. Artiﬁcial insemination at approximately 36 h before the time of ovulation will
increase the percentage of female calves.
2. Uterine horn artiﬁcial insemination will attenuate the decrease in CR in lactating

dairy cows when inseminated approximately 36 h before ovulation.

Chapter 1

REVIEW OF LITERATURE

Timing of Artiﬁcial Insemination, Site of Semen Deposition and Management Factors

that Inﬂuence Fertility in Lactating Dairy Cows and Gender Ratio of Offspring

Part 1: Timing of Artiﬁcial Insemination (AI )
Optimal Time of AI in Relation to Estrus or Ovulation

During the development and validation of AI, several studies investigated the
optimal time of AI in relation to estrus to achieve highest fertility (Aschbacher et al.,
1956; Barrett and Casida, 1946; Hall et al., 1959; Trimberger, 1948; Trimberger and
Davis, 1943). Optimal fertility was reached from mid-estrus until a few hours after the
end of estrus. These data were responsible for the establishment of the am-pm or 12 h
rule (if a cow is in estrus in the morning then AI in the evening). Later work suggested
that inseminations during the middle to the end of estrus resulted in higher fertility than
early estrus (Fields et al., 1975; Hall et al., 1959). Robbins et al. (1978) found that beef
cows inseminated within 24 h following ﬁrst observation of estrus did not have a
reduction in fertility. However, cows inseminated more than 24 h after onset of estrus
had a greater percent of degenerate embryos on d 6 or 7 (Dalton etal., 2001).

The variability in the interval between onset of estrus and the actual observation
of estrus made it difﬁcult to deﬁne the ideal time of AI in relation to ovulation (F oote,
1979; Gwazdauskas et al., 1986). However, development of Ovsynch (Figure 1)
provided an effective protocol for determining the optimal time of AI (Pursley et al.,
(1995). Ovsynch effectively controls the time of ovulation, thus allowing for precise
timing of AI in relation to ovulation. In two studies, greater than 95% of cows ovulated a
dominant follicle 24 to 32 h after the ﬁnal GnRH injection. This is a dramatic reduction
in the variability of time to ovulation (8 h) compared to previous synchronization

programs using PGan in which the range of time to ovulation was 5 d (Pursley et al.,

1997).

 

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Figure l. The timing and purpose of hormone injections in the Ovsynch program, adapted
from (Pursley etal., 1995).

Pursley et al. (1998) showed that similar PR could be achieved following an Ovsynch
program, with inseminations occurring at 0, 8, 16, and 24 h aﬁer ﬁnal injection of GnRH.
Yet, cows inseminated 16 h following GnRH of Ovsynch had the highest fertility,
whereas cows in the 0 h group had the lowest embryonic loss. Interestingly, the 0 h
group (inseminated approximately 24 to 32 h prior to ovulation) had the 2nd lowest CR

but produced more female calves.

Importance of Increasing Female Ojfspring

The average female: male gender ratio in dairy calves born in the 19705 was 49:
51 (Foote, 1977). However, more current studies with large sample sizes, had outcomes
of approximately 46: 54 females: males (Berry et al., 1995; Ryan and Boland, 1991). A
lower percentage of heifers born poses a challenge because number of heifer calves
raised must equal the number of cows culled to maintain herd size. Currently, the
average culling rate in 12,000 herds (DRMS, Dairy Metrics June 2003) was
approximately 39 % per year. In the US. total number of cows is slowly declining. This
would insinuate there is insufﬁcient numbers of heifers to replace the cows that are being

culled. There are two types of culling, voluntary and involuntary. Voluntary culling

consists of cows leaving the herd because of a management decision. Involuntary culling
is an unavoidable culling decision based on the health of the cow (Weigel et al., 2002).
Involuntary culling of once productive cows reduces farm proﬁtability and leads to the
need for more replacement females. Increasing the number of replacement heifers would
alleviate the pressure of both voluntary and involuntary culling of dairy cows.

Predetermining the gender of mammalian offspring with sperm sorting or embryo
sexing is not a new idea (Bondioli et al., 1989; Pinkel et al., 1982). Johnson et al. (1989)
produced the ﬁrst live rabbit offspring using a sperm sorting technique. Rabbits
inseminated with X-bearing sorted sperm had 94 % females. Since then, offspring of
preselected sex have been produced with 90 % accuracy in pigs, cattle, sheep and most
recently horses (Buchanan et al., 2000; Cran and Johnson, 1996; Seidel et al., 1999).
However, sperm sorting has not become commercially accepted because of expensive
equipment required, slow processing rates, and reduced sperm quality.

Timing of AI in relation to ovulation is a comprehensible and economical method
for altering gender. Work in cattle indicated that timing of insemination can alter gender
ratio. Pursley et al. (1998), and Vasconcelos et al. (1997) altered the gender ratio in favor
of females when insemination occurred ~ 28 h prior to ovulation in lactating dairy cows.
However, CR was reduced approximately 20 %. A recent in vitro fertilization (IVF)
study tested whether sperm incubation prior to oocyte fertilization affected sex ratio of
blastocysts. Prolonged incubation of sperm for 24 h prior to fertilization reduced
development and fertilization rates, but more female-hatched blastocysts were present on
d 9 (Lechniak et al., 2003). However, Rorie et a1. (1999) published data that conﬂicts

with the previously described work. His work was based on time of insemination at

either 20 h before ovulation or 10 h after ovulation. No difference in gender ratio of
calves born was found. Twenty hours may not be sufﬁcient enough time for X and Y
chromosome-bearing sperm to deviate. Additionally, small sample size or poor

prediction of time of ovulation may explain the conﬂicting results.

Sperm Survival in the Female Reproductive Tract

Timing of AI that occurs farther from the time of ovulation may reduce sperm
survivability and CR. Spermatozoa survivability in inseminated cows was tested as early
as 1945 and determined that sperm survive for only 30 h (Laing, 1945). However, it was
reported later that bovine sperm have a fertile life of 30 to 48 h in the uterus (Bazer et al.,
1987). Sperm adherence to the epithelial lining of the ampulla extends sperm longevity.
Although sperm can survive in the female reproductive tract for 2 d, fertility is reduced.
Optimal sperm survival rate and subsequent fertility may be dependent on timing of

insemination relative to time of ovulation.

Semen Quality and Quantity

Quality and quantity of sperm present at the site of fertilization following
prolonged exposure in the uterus may also affect fertility. A number of studies have
investigated effects of quality and quantity of the semen using traditional AI practices
(insemination 12 h aﬁer observed estrus). The optimal sperm number to achieve
maximal fertility varies among bulls. Therefore, AI companies use relatively high

numbers of sperm (approximately 20 million) to compensate (Foote and Parks, 1993).

Bull fertility differences can be divided into two categories. Compensable traits,
which can be compensated with semen quantity, or uncompensable traits, which cannot
be compensated with increased quantity (Saacke et al., 2000). The number of accessory
spermatozoa, fertility rate and embryo quality increase when sperm per insemination dose
increased from 20x106 to 100x106 madir etal., 1992). Accessory sperm number present
in the zona pellucida of the oocyte increased from 3 to 27 (Saacke et al., 1994).
Accessory sperm are sperm trapped in the zona pellucida by the zona reaction after
penetration of the fertilizing spermatozoon. The authors suggested that quantity of sperm
might stimulate competition among fertilizing sperm, which may be favorable to
embryonic development as well as fertilization rates. A possible mechanism for this
hypothesis is a greater number of sperm at the site of fertilization may include more
highly competent sperm. Another study considered 75 d non-retum rates using different
semen extenders with various semen doses. Results indicated that low semen dose
caused decreased fertility (Schenk et al., 1987). denDaas etal., (1998) found that sperm
numbers needed to obtain 95 % of the maximal CR in dairy bulls ranged from 1 to 1 1
million. Different semen extenders in this study did not differ signiﬁcantly. Taken
together this literature indicates that a certain minimum number of sperm are necessary
before fertilization can occur.

Sperm characteristics related to quality are motility, membrane integrity,
acrosome integrity, and ability to bind the zona pellucida (den Daas, 1992). Successful
penetration of the oocyte depends on quality of the sperm inseminated. This is also
known as extrinsic sperm quality. Intrinsic quality is the paternal contribution to proper

development of the zygote. This is dependent on the quality of sperm (den Daas, 1992;

Pace et al., 1981). Cows inseminated with above average quality semen had 21% more
number 1 and 2 quality embryos and 27% fewer degenerated embryos and unfertilized
ova than cows inseminated with below average semen (Nadir et al., 1992). Beef heifers
inseminated with either high or low fertility semen had a fertilization rate of 89 % and 70
% respectively (Callaghan and King, 1980). Call and Stevenson (1985) suggested that
greater emphasis should be placed on selection of genetically superior sires for both milk
production and fertility.

Clay and McDaniel (2001) developed a model for calculating relative bull fertility
called the Estimated Relative Conception Rate (ERCR). The ERCR provides producers a
management tool for selecting high fertility bulls. Selection of higher fertility bulls
could lead to improved reproductive efﬁciency. ERCR measures the 70 d non-return rate
of AI service sires relative to other service sires used within the same herd. ERCR is
predicted using only ﬁrst service insemination data and only after a bull has had at least
300 inseminations. Environmental factors such as herd, month of insemination, parity of
cow, days in milk (DIM) and milk production are taken into account when calculating
ERCR. A bull with a ERCR of 4, means that the bull should have CR 4 % greater than
contemporaries. Bulls with higher CR potential may give producers a viable tool to

increase reproductive efﬁciency.

Sperm Capacitation and Binding
Capacitation is deﬁned as the changes sperm undergo within the female
reproductive tract to achieve fertilization capability. Timing of AI may affect the ability

of sperm to become capacitated. Capacitation activities peak in bovine oviductal ﬂuid

10

around the time of estrus and decline during the luteal phase of the estrous cycle (Didion
and Graves, 1986; Lauderdale and Ericsson, 1970). Zona binding proteins on the sperm
head must be exposed during capacitation in order for ZP3 binding to occur (Senger,
1999). To determine the exposure time needed to capacitate sperm, Lauderdale and
Ericsson (1970) labeled bull semen with tetracycline HCl (T-HCl) and placed it in uteri
of cows and heifers. The absence of ﬂuorescence was an indirect method to detect
capacitation. Sixty percent of sperm recovered from females in estrus had capacitated
sperm 5 h aﬁer AI. Only 2% of cows in the luteal stage of the estrous cycle had
capacitated sperm. Didion and Graves (1986) supported these ﬁndings and also analyzed
sites within the female reproductive tract that might differ in ability to affect capacitation.
Most of the capacitated sperm was found in the ampulla, ipsilateral to the dominant
follicle.

Sperm binding to the zona pellucida is the ﬁrst gamete interaction in the
fertilization process and lack of interaction may be a common defect in some bulls
(Braundmeier et al., 2002). Later steps in fertilization such as zona penetration,
membrane fusion, or oocyte activation, or even an earlier step prior to zona binding, may
also contribute to reduced fertility.

Certain regions of the female reproductive tract regulate sperm function as they
progress through the oviduct (Boquest et al., 1999). Oviductal proteins may extend the
longevity of spermatozoa during preovulatory periods and promote the acrosomal
reaction (Boquest et al., 1999). The acrosome reaction begins when the plasma
membrane fuses with the acrosomal vesicle membrane. Without this reaction sperm

cannot penetrate the oocyte. The acrosome reaction allows penetration of the oocyte by

11

releasing enzymes on the sperm head. This allows binding and digestion of the zona
pellucida to occur. Boquest et al., (1999) found that proteins from bovine oviductal
epithelium promote sperm viability, delay acrosome damage, and suppress sperm
locomotion. Possible changes in osmotic pressure and temperature could cause
irreversible damage to the sperm acrosome, and diminish sperm fertilizing capability

(Senger, 1999).

Summary

AI 12 h following initial observation of estrus once was the optimal time to
achieve greatest fertility. With the advent of Ovsynch, timing of AI in relation to
ovulation is possible. Pursley etal., (1998) determined that the optimal insemination
time to achieve highest fertility is 16 h following the LH surge (ﬁnal GnRH injection of
GnRH) of Ovsynch. Ovsynch allows precise timing of ovulation approximately 24 to 32
h following the LH surge. AI ~ 28 h before ovulation produces more female offspring
than A1 at 16 h before ovulation. This is particularly important to dairy producers, whose
livelihood depends on replacement females. However, AI 28 h prior to ovulation is not a
viable management practices because fertility is decreased. The decrease in fertility may
be due to semen quality or quantity inseminated, or the ability for sperm to survive,

capacitate and remain fertile in the female tract prior to fertilization of the oocyte.
Part 11: Site of Semen Deposition

Determining the ideal site of semen deposition to gain greatest fertility is

important to AI efﬁciency. In a review by (Lopez-Gatius, 2000), the author reported that

12

vaginal insemination was replaced with cervical insemination some time in the 19403.
Then in the 19503 and 19603, experiments using fresh semen indicated little difference in
fertility rates among the uterine horn, uterine body or mid-cervix insemination (Knight et
al., 1951; Macpherson, 1968; Olds et al., 1953). However, Weeth and Herman (1951)
indicated that cows inseminated in the uterine body had higher fertility than cows
inseminated in the cervix. Furthermore, Macpherson (1968) used high concentrations of
frozen-thawed sperm and determined that placement of semen in the uterine body instead
of the cervix increased cow fertility. These data were responsible for establishment of
current A1 procedures. However, with the decline in fertility in lactating dairy cows,
reproductive specialists are re-investigating site of semen deposition to achieve greater
fertility. Presently, pregnancy following a single AI in lactating dairy cows rarely results
in rates higher than 40 % (Pursley et al., 1997; Washbum etal., 2001). This is alarming
considering that in the 19605, it was common to have PR of 60 % or better (Spalding et
al., 1974).

Two of the primary arguments for use of uterine horn insemination are: placement
of semen nearer to the uterotubal junction (main sperm reservoir prior to ovulation) and
reduction of incidental cervical deposition (Lopez-Gatius, 2000). There are two types of
uterine horn insemination methods, bicomual and unicomual. Senger et al. (1988)
demonstrated a dramatic increase in CR when using bicomual insemination compared
with uterine body insemination. CR on 4000 cows were improved from 45 to 70 % when
cows were inseminated by splitting the semen in one straw between each horn compared
with the standard deposition of one straw in the body of the uterus (Senger et al., 1988).

However, other investigators have failed to conﬁrm above results (Graves et al., 1991;

13

McKenna et al., 1990; Williams etal., 1988). More recent work of Pursley and co-
workers (2003; unpublished) demonstrated increased CR in lactating dairy cows (from 35
to 50 %) with deep uterine horn AI (11 = 800) as compared with uterine body. In addition,
Diskin and Pursley (2003; unpublished) observed increased fertility in 3200 lactating
dairy cows in Ireland (from 45 to 50 %) using deep uterine horn insemination.

The unicomual insemination method deposits semen in the horn either ipsilateral
or contralateral to the side of ovulation. As expected, higher fertility was observed in
cows inseminated in the ipsilateral versus the contralateral horn (Dalton et al., 1999;
Lopez-Gatius, 1996; Lopez-Gatius and Camon-Urgel, 1988; Zavos et al., 1985). Hawk
and Tanabe (1986) and Momont et al. (1989) found no difference in fertilization or PR
when uterine body insemination was compared with horn insemination ipsilateral to the
side of impending ovulation.

The contradictory results among horn insemination studies may be attributed, in
part, to various insemination depths and techniques of deposition within the horn, as well
as inaccuracy of placement of semen in the uterine horn or uterine body. lnaccuracy of
semen placement within the reproductive tract was ﬁrst observed following a dye
deposition technique (Wright, 1964). Years later, a more sophisticated radiographic
technique was established to determine proper position of the syringe tip in the uterine
body. It was established that inseminators failed 61 % of 586 attempts to correctly
deposit semen in the uterine body and more than 20 % of those attempts were in the

cervix (Peters et al., 1984).

14

Sperm Transport in the Female

Site of semen deposition in the female reproductive tract may have an impact on
sperm transport. Sperm deposited in the uterine body become widely dispersed
throughout the reproductive tract, in a forward movement. Sperm are transported more
efﬁciently in animals mated near the time of ovulation than those mated early in estrus
due to uterine contractions and mucus ﬂow (Hunter and Wilmut, 1983). Normal sperm
transport in the female takes place in two phases: 1) Rapid transport phase in which
sperm reach the oviduct within a matter of minutes, but do not have fertilization
capability; 2) Sustained transport phase, where sperm capable of fertilizing ova reach the
oviduct about 8 h after insemination (Wilmut and Hunter, 1984). Hunter and Wilmut
established that sperm capable of fertilization were present in the oviduct 10-12 h after
insemination. Sperm were held in the caudal end of the oviduct (isthmus) 18 h or more
until ovulation at which point sperm proceeded toward the site of fertilization (Hunter
and Nichol, 1986). Early research regarded the cervix as a sperm reservoir prior to
ovulation (Mattner, 1966; Mattner, 1968). More recent work by Hawk (1987) and Hunter
and Wilmut (1983) indicated that the isthmus is the primary pre-ovulatory reservoir at the
time of ovulation. Thus, deposition of semen in the uterine horn would bring sperm
closer to the pre-ovulatory reservoir. Lowered temperature within the isthmus of the
oviduct at estrus reduces sperm motility, which allows sperm to reside longer in this
reservoir prior to fertilization (Hunter and Nichol, 1986).

The effectiveness of sperm transport can be measured directly or indirectly
(Hawk, 1983). An indirect measure of sperm transport is cow fertility. Counting sperm

in various sections of the reproductive tract following insemination can be used as a

15

direct measure of sperm transport. Numerous investigators have used this direct measure
of sperm transport (el-Banna and Hafez, 1970; Hafez and Dobrowolski, 1970; Hunter and
Wilmut, 1983; Mitchell etal., 1985) to determine the fate of sperm in the female
reproductive tract following AI. Direct measure of sperm transport has allowed
investigators to determine that sperm capable of fertilization are present in the oviduct
10-12 h after insemination (Wilmut and Hunter, 1984). Other direct measures of sperm
transport are fertilization rate and number of accessory sperm attached to the zona
pellucida. However, there is controversy over whether accessory sperm represent
increased fertility. Hawk and Tanabe (1986) determined that the number of accessory
sperm in the zona pellucida following uterine horn insemination did not necessarily
increase fertility. They found that zygotes developed with or without accessory sperm
present. In contrast, Dalton et al. (1999) indicated that unicomual insemination provided

greater sperm transport to the oocyte because of the number of accessory sperm present.

Uterine Environment and Fertility

Morrow et al. (1969) reported that slow involution of the uterus after calving was
associated with inefﬁcient reproduction. Uterine horn deposition may mitigate the effects
of inferior uterine involution on sperm transport and fertility. Health of the uterine,
cervical and oviductal environments are crucial to sperm transport and maintenance of a
successful pregnancy (Lopez-Gatius, 1996). Lopez-Gatius investigated the effect of
uterine horn insemination (ﬁrst parity cows) in either the previously non gravid horn or
gravid horn (pregnant horn) on sperm transport and PR. The investigators found that

deposition of semen into the previously non-gravid horn increased PR when compared

16

with previously gravid horn insemination. This would indicate that previous pregnancy

may negatively inﬂuence future reproductive efﬁciency.

Pitfalls of Uterine Horn Insemination

Although uterine horn insemination may increase CR, this technology has not
been readily adopted by the dairy industry. One of the major concerns with uterine horn
insemination is damage to the endometrium and introduction of bacteria. Larsson et al.
(1986) discovered uterine horn insemination caused minor hemorrhage in the mucus
membrane of heifers 2 d after insemination. Williams et al. (1987) addressed the effects
of uterine horn insemination on health of the reproductive tract. Reproductive tracts were
harvested 5 d after AI and evaluated for discoloration and luminal bacterial count.
Neither uterine body nor comual insemination increased bacterial population or trauma to
the uterus. Either the uterine lumen was not damaged from Al or it had the capability to
repair damage and eliminate bacteria within 5 d after A].

In addition it was once believed that uterine horn insemination was very difﬁculty
to teach and hard to execute. However, researchers have devised ways to train effective
inseminators. McKenna et al. (1990) and Williams et al. (1988) trained inseminators on
excised tracts using dye that stained the uterine lining at the site of deposition.
Additionally, Bea] et al. (1991) used an ultrasound to determine the location of a metal
swivel attached to ﬁshing line deposited into the uterine horn. These teaching procedures
were 90 % effective in training inseminators to accurately place semen deep in the uterus

(Pursley et al., 2003; unpublished).

17

Summary

Site of semen deposition to gain the greatest fertility has been investigated for
many years. The most widely accepted site of insemination is the uterine body. The
disadvantage to this site is increased incidence of cervical deposition. Uterine horn
deposition is an alternative to uterine body insemination and is recommended as a way to
limit cervical insemination. Additionally, uterine horn deposition may serve as a
technique to by-pass uterine environments that hinder sperm transport and promote sperm
loss. Skeptics argue that no advantage in fertility is gained when uterine horn
insemination is used. Furthermore, concerns regarding damage to the uterus have been
raised. However, the uterus seems to have the ability to repair any damage within 5 (1

following AI.

Part III: Physiological and Management Factors that may Affect Fertility

Efforts by dairy producers to remain proﬁtable over the last two decades have
resulted in increased herd size and more intense management practices. The increased
demand on dairies means an increased demand on cows, and particularly production
performance. It is important that dairy producers are aware of physiological and
management factors that may reduce fertility and carefully manage cows to obtain higher
fertility (Britt, 1985; F onseca et al., 1983; Hillers et al., 1984; Oltenacu et al., 1984).
Weigel (2002) reported that 96% of the variation in CR in lactating dairy cows is related
to management and environmental factors. The following are some physiological and

management factors that may affect cow fertility.

18

Genetics

Cow fertility is less than 3 % heritable (Hansen, 2000). Genetic control of cow
fertility is extremely low, making environmental and management factors important to
fertility (Hansen, 2000). Many studies attribute single trait selection for high milk
producing cows as the cause for decreased fertility. Hansen (2000) suggests that in order
for cows to become more reproductive efﬁcient in the future, selection of traits that affect
the longevity such as cow conformation and overall performance must be considered. In
addition, genetic selection of bull fertility may offer the greatest opportunity to improve

conception rates in lactating dairy cows.

Stress

The most important effect of stress on reproduction is disturbance of
hypothalamic function. Acute stressors reduce normal pulsatile patterns of GnRH and
LH (Dobson et al., 2001). Mastitis is one example of stress that may reduce fertility in
cows. Infection of the udder can cause pain and stress for a cow, particularly severe
cases. Somatic cell counts (SCC) are routinely used as an indicator of subclinical
mastitis. Cows with high SCC have longer calving intervals than normal cows (Dobson
etaL,2001)

Environmental heat stress is another major stressor for cows. Cows subjected to
heat stress during the meiotic maturation process have increased numbers of abnormal or
poor oocytes (Putney et al., 1989). Two experiments in two seasons evaluated
fertilization rates and embryonic development in lactating and non-lactating dairy cows

(Sartori et al., 2002). Experiment 1 (summer) compared lactating Holstein cows to

19

nulliparous heifers. Experiment 2 (winter) compared lactating cows to dry cows.
Fertilization rates were reduced only in summer in lactating cows, which the author
attributed to heat stress (Sartori et al., 2002). Embryos allocated from of lactating dairy
cows in summer had a greater percentage of grades 3, 4 and 5 embryos (low quality) than
non-lactating females. In winter, fertilization rates were similar in lactating and non-
lactating cows.

Heat stress has a negative effect on oocyte quality both in vivo and in vitro,
particularly in lactating dairy cows. Lactating dairy cows have higher body temperatures
than non-lactating animals in response to increasing environmental temperature. This
could be due to higher metabolic energy associated with feed intake and milk production,
which makes them more prone to heat stress (Berman etal., 1985). Rivera and Hansen
(2001) found that in vitro exposure of oocytes and sperm to 41° C during fertilization
reduced the number of zygotes that cleaved to two cells. Lenz et al. (1983) also found
that exposure of oocytes to high temperatures decreased oocyte maturation and

subsequent fertilization rates.

Parity

Older cows have more reproductive disorders and lower reproductive
performance (Hillers et al., 1984; Lerner et al., 1986). Cows in third and greater
lactations had lower CR and longer intervals to ﬁrst service than primiparous cows
(Coleman et al., 1985; Hillers et al., 1984). The possibilities of more periparturiant
diseases that occur with more calvings could be one possible explanation for lowered

fertility (Erb and Martin, 1980).

20

CR from A1 in ﬁrst calf dairy heifers have remained close to 70 % over the past
10 years, whereas fertility in multiparous lactating cows has steadily declined (Foote,
1975; Pursley et al., 1997; Spalding et al., 1974). Given that heifers are often housed and
fed a similar diet as cows, one could speculate that calving and (or) lactation may have
some effect on cow physiology that reduces fertility. The condition of the uterus at
parturition, or soon thereafter, is a major reason for low fertility in lactating dairy cows

(Erb and Martin, 1980; Fonseca et al., 1983; Kay, 1978).

Retrograde Sperm Loss

Retrograde sperm loss is sperm discharged from the female tract. The effect of
parturition on the uterus and retrograde sperm loss in dairy cows is unclear. However in
non-lactating heifers we know the majority of retrograde sperm loss occurs 2 h following
AI (Gallagher and Senger, 1989). Mitchell et al. (1985) recovered approximately 60 %
of sperm inseminated in the mucus expelled from the vagina of multiparious dairy cows.
The author also reported that retrograde sperm loss helped reduce the number of
abnormal sperm present in the reproductive tract (Mitchell et al., 1985). Gallagher and
Senger (1989) reported approximately 20 % of sperm were lost following insemination in
the uterine body or horn in heifers. Although data do not exist that directly compare
heifers with lactating cows for retrograde sperm loss, 1 speculate that sperm loss is
greater in cows than heifers. This could be due to difference in the size and the
physiological status of the uterus between cows and heifers. This may partially explain

the difference in CR between dairy heifers and lactating dairy cows.

21

Phagocytosis

Phagocytosis is another way the female rids the reproductive tract of sperm. The
presence of sperm in the female genital tract increases the number of leukocytes in the
lumen of the uterus and cervix (Mattner, 1968). Sperm are foreign to the uterus and
neutrophils attack “non-self" material upon entrance (Mattner, 1969).

During estrus when the uterine epithelium is under the inﬂuence of estradiol,
leukocytes (speciﬁcally neutrophils) accumulate in the mucosa of the vagina and uterus
(Senger, 1999). Mitchell et al. (1985) speculated that about 27 % of sperm loss after
insemination was due to phagocytosis. However, this number may increase when estrus
synchronization is used. Estrus synchronization may cause a “hyper-estrogenic”
condition in cattle that triggers increased leukocytic activity, speeding up capacitation
and phagocytosis of sperm (Lauderdale and Ericsson, 1970). Increased phagocytosis in
lactating dairy cows may reduce number of sperm at the site of fertilization, thus

reducing fertility.

Locomotion Scoring

Locomotion scoring is a method to assess the amount of lameness present in a
herd. Lameness negatively affects the ability of cows to express estrus. Herds with
prevalent lameness experience a decline in both the rate and accuracy of detection of
estrus (Sprecher et al., 1996). Senger ( 1994) suggested that detection of estrus is the
single most important limiting factor for high reproductive efﬁciency. Lameness can also
lower ﬁrst service CR and increase incidence of ovarian cysts (Melendez et al., 2003).

Hernandez et al. (2001) found that the type of foot disorder impacted fertility. Cows with

22

abscesses/sole ulcers had 63 more days open than healthy cows. Cows with two or more

foot disorders had 76 more days open when compared with healthy cows.

Body Condition Scores and Nutrition

Energy requirements of lactating dairy cows are met through a combination of
dietary intake and mobilization of body reserves. Cows producing large quantities of
milk cannot consume enough feed to maintain a positive dietary energy balance. During
early lactation the cow must mobilize body stores to accommodate metabolic
requirements (Nebel and McGillard, 1993). Body condition scoring (BCS) (based on a 5
point scale) is a method to evaluate the amount of body fatness a cows maintains
(Wildman et al., 1982). BCS can be used to adjust dairy herd rations and herd health
programs to achieve higher cow productivity, fertility, and longevity. Pulsatile LH
secretion is suppressed, until negative energy balance reaches the lowest level and begins
returning towards a positive energy balance (Butler and Smith, 1989; Canﬁeld and
Butler, 1990). Once energy balance is reached, LH secretion is reestablished and
ovulation occurs. Body condition early in lactation is one of the best indicators for
potential anestrous in a cow. Additionally, cows that are over or under-conditioned are
more susceptible to metabolic problems, infections and reproductive difﬁculties
(Gearhart et al., 1990). Cows with a BCS of 3.0 (on a 5 point scale) at Al may have the
greatest likelihood for pregnancy success (Loefﬂer et al., 1999). In contrast, Waltner et
al. (1993) indicated that BCS had no signiﬁcant relationship to incidences of pyometra,
metritis, RP, cystic ovaries or dystocia. However, the author cautions over interpretation

of the data, as the study was conducted on one farm with little variation among BCS.

23

Herds with more variation in BCS may exhibit a signiﬁcant relationship between BCS
and reproductive diseases.

Proper nutrition is essential for energy balance. This balance is crucial to
follicular growth, oocyte quality, embryo sun/ival and overall fertility in the lactating
dairy cow (O'Callaghan and Boland, 1999). Numerous studies of the nutritional needs
and fertility in high producing cows have been published (Studer, 1998). Britt (1977)
noted that mistakes in feeding dairy cows in late lactation and during the dry period
caused the most damage to reproductive success. This coincides with greater embryo
loss in dairy cows that have low or high BCS in early gestation (Lopez-Gatius et al.,
2002; Silke et al., 2002). A recent review of nutritional effects on ovulation, embryo
development and establishment of pregnancy in ruminants collectively indicated
deleterious effects of high dietary intake and excess crude protein on fertility
(O'Callaghan and Boland, 1999). The lack or overuse of vitamins and minerals was also
detrimental to fertility (Swanson, 1989). The effects of nutrition can be found in early
oogenesis even before the oocyte develops competency (Boland et al., 2001). Boland et
al. (2001) reviewed effects of nutrition on follicular development, oocyte quality and
embryonic development in ruminants. High dietary intake exerts a negative effect on
developmental capacity of embryos. This may be due to increased blood ﬂow and steroid
metabolism in lactating dairy cows. (Butler and Smith, 1989; Sangsritavong et al., 2002;

Wiltbank et al., 2000).

24

Bovine Somatotropin

Bovine somatotropin (bST) is a growth hormone in cattle. Administration of bST
to lactating dairy cows increases milk production (Peel and Bauman, 1987). Producers
that use bST can expect an 8 to 17 % increase in milk production. However, there are
disadvantages to using bST. Cows treated with bST may have decreased energy balance
(Lucy etal., 1993). Also, treated cows do not express estrus as intensely as untreated
cows (Kirby et al., 1997). Therefore, bST-treated cows may have fewer opportunities to
become pregnant.

Dalton and Marcinkowski (1993) evaluated the effect of bST on steroid hormones
in the female and indicated no effects on basal GnRH during the breeding period. Yet,
Kirby et a1. (1997) reported that cows treated with bST had reduced F SH, and a faster
turnover of dominant follicles. Other research also indicated that bST reduced FSH but
increased initial development of the CL and extended its functional life span (Lucy et al.,
1994)

A study was conducted to evaluate long-term effects of bST on fertility. Data
from cows treated with bST over a 2-year period were collected. Multiparous cows
treated with bST for 1 year had increased days to conception. The cows treated with bST
for 2 years tended to have lower CR than control cows (71 vs 87% ; (Hansen et al.,
1994). Additionally, increasing dose of bST decreased body condition linearly at the end
of both years. Cows in their second year of treatment were slower to regain body
condition. Another study reported that cows treated with bST had a signiﬁcantly lower
PR at 180 DIM than controls (Luna-Dominguez et al., 2000). In contrast, bST increased

PR after a ﬁrst Ovsynch synchronized insemination (Moreira et al., 2000). Given the

25

varied results of bST on fertility parameters, more research is needed to understand the

true impact of bST on fertility.

Retained Placenta and Dystocia

Retained placenta (RP) is generally deﬁned as failure to expel the placenta within
8 to 12 h after calving (van Werven et al., 1992). However, other work has deﬁned RP as
the failure to expel within 24 h and even up to 48 h after parturition (Halpem etal., 1985;
Lee et al., 1989). Twins, abortions, and stillbirths have been associated with higher
incidence of RP (Youngquist and Bierschwal, 1984). It is difﬁcult to evaluate the effect
of RP on fertility because there is no set standard for normal expulsion of the placenta.
vanWerven (1992) studied the effect of RP retention time on fertility. RP did not alter
reproductive performance nor culling rate of ﬁrst, second or third lactation cows.
However, cows in fourth or greater lactation had lower fertility as duration of RP
increased.

A primary concern in cows with RP is increased susceptibility to uterine
infections (Erb and Martin, 1980). Holt et al. (1989) investigated the effect of GnRH
injection at 15 d postpartum in cows that were clinically normal versus cows with uterine
discharge or RP (> 24 h). Effects on ovarian function, progesterone concentrations and
overall fertility were examined. The results indicate that cows with RP had the lowest
CR and most days to ﬁrst service. Loefﬂer et al. (1999) pointed out that cows with
cloudy uterine discharge after calving were only half as likely to conceive as females

with a clear discharge.

26

Dystocia is deﬁned as delayed and difﬁcult parturition (Berger, 1994). Many
producers use a calving difﬁculty score to quantify (on a l to 5 scale) difﬁculty of birth.
Calving difﬁculty in Holsteins can impair subsequent reproductive performance by
increasing days open, and increasing services per pregnancy (Mangurkar et al., 1984;
Thompson et al., 1982; Thompson et al., 1983). Possible losses attributed to dystocia
include reduced milk production and fertility, death of cow or calf and increased

veterinary costs (Dematawewa and Berger, 1997).

Twinning

Multiple births are desirable in many species. However, birth of twins in dairy
cows is associated with increased dystocia and health problems for the cow such as RP
and uterine infections. Additionally, cows with twins have about 22 more days open
than cows with singletons (Chapin and Van Vleck, 1980). Jones and Rouse (1920)
associated twinning with older cows. Cole and Rodolfo (1924) associated twinning to
time of year, and Lush (1925) to genetics. More recent work attributes increased
twinning rates to increased parity and milk production (Kay, 1978; Wiltbank et al., 2000;
Wood, 1975; Wood, 1984).

Most twin calves occur as a result of multiple ovulations (dizygotic twins)
(Wiltbank et al., 2000). Wiltbank et al. (2000) suggested that multiple ovulations in high
producing dairy cows are due to increased intake of dry matter. Increased dry matter
intake increases liver blood ﬂow and steroid metabolism. Normal dominant follicle
selection usually results in an increase in circulating of estradiol, which suppresses

follicle stimulating hormone (FSH). However, with increased estradiol metabolism the

27

concentration of F SH remains higher, allowing a second follicle to be recruited into
dominant status. Therefore, higher producing cows may be more prone to double
ovulations than lower producing cows (Fricke and Wiltbank, 1999). Wiltbank et al.
(2000) reported that 20 % of high producing cows have double ovulations whereas only 7

% of low producing cows have double ovulations.

Milk Production, Days in Milk and Milking Frequency

Extensive research has been dedicated to understanding the relationship between
milk production and fertility. Indeed, most studies reported that high milk production
(herd average) had a negative effect on fertility (Faust et al., 1988; F onseca et al., 1983;
Laben et al., 1982; Ron etal., 1984; Spalding et al., 1974) while others found no effect or
slightly positive results (Boyd et al., 1954; Everett et al., 1966; Gaines, 1927; Hillers et
al., 1984). Some studies indicated that good management of metabolic needs of high
producing cows could overcome the negative effect of milk production on fertility (Call
and Stevenson, 1985; Laben et al., 1982; Nebel and McGillard, 1993). Call and
Stevenson (1985) found (from DHIA herd summaries) that increased rolling herd
averages were accompanied by an increased interval to ﬁrst service and number of open
cows. Hillers et al. (1984) examined the inﬂuence of management and environmental
factors on reproduction in four large Washington dairy herds. Milk yield did not affect
ﬁrst service CR. However, others reported that cows with high milk yield had longer
intervals to ﬁrst service (Call and Stevenson, 1985). Other work indicates that selection
for milk yield improved fertility in virgin heifers (Hansen etal., 1983). Recently, cows

with above-average milk production within one high producing herd had greater PR/AI

28

(45.8 vs. 33.8%) compared with below-average herdmates (Peters and Pursley, 2002).
An explanation for high fertility in higher producing cows may be that they have fewer
health problems, such as lameness, mastitis, RP, or ketosis. The observation that poor-
milk producing cows have more health problems and are less likely to conceive (Badinga
et al., 1985) supports this explanation.

Days in milk (DIM) may also affect fertility. Loefﬂer et al. (1999) found a
lowered pregnancy success in both early and late lactation females. Dairy cows achieved
optimal fertility at approximately 82 d after parturition. This time following parturition
allows cow to reach a positive energy balance and time for their uteri to properly
involute. In addition to days in milk, milking frequency may also affect fertility in
lactating dairy cows. A 15—16 % increase in milk production can be expected when
milking frequency is increased. It has been documented that there are longer calving
intervals and more services per pregnancy in cows milked 3 times per day (3X) than 2X

(Smith et al., 2002).

Summary

Management practices that emphasize genetic selection for healthy efﬁcient cows
with good locomotion and body condition score may enhance reproductive efﬁciency.
Retrograde sperm loss and phagocytosis are responsible for signiﬁcant amounts of sperm
lost from the reproductive tract following AI. Increased sperm loss may mean lowered
numbers of sperm at the site of fertilization and thus lowered fertility. This may be
confounded by age of cow. Older cows experience more reproductive disorders and

lower fertility. Monitoring and managing mastitis, heat stress, and postpartum uterine

29

health may be beneﬁcial to lessen the effects of parity, retrograde sperm loss and
phagocytosis on fertility in the lactating dairy cow. The relationship of increased milk

production and use of bST on the decline in fertility remains unclear.

30

Chapter 2

PAPER TO BE SUBMITTED TO JOURNAL OF DAIRY SCIENCE

Effect of Timing of Artiﬁcial Insemination and Placement of Semen on Fertility in

Lactating Dairy Cows and Gender Ratio of Resulting Offspring

M.W. Macfarlane, G.J.M. Rosa, J .R. Pursley
Department of Animal Science

Michigan State University

31

MATERIALS AND METHODS

This study was conducted on two dairy farms in Michigan and one in Kansas.
Primiparous and multiparous lactating dairy cows (n = 1603) were randomly assigned to
one of four treatments based on lactation number (1, 2, 2 3) and DIM (< 150 and 2 150
DIM). Treatments were arranged as a 2 x 2 factorial experiment as illustrated in Table 1.
All farms maintained cows in free-stall barns with sand bedding and headlocks. Research
began March 2002 at Farm 1 (n = 133) located in mid Michigan (700 lactating cows)

with a rolling herd average of 1 1,298 kg and ended May 2002. Cows were milked

Table 1. Sample size for each of the four treatment groups.

 

Tune of insemimtion relative to the ﬁnal GnRH of Ovsynch

 

Site of

 

. . -8 h 16 h
Insemination
Body Group 1 (n=400) Group 2 (n=417)
Horn Group 3 (n=372) Group 4 (n=414)

 

twice daily for the majority of the herd and four times per day for cows early in lactation
(70 to 80 DIM for 2-yr olds and 50 to 60 DIM for mature cows). Cows were fed a corn
and alfalfa silage based ration balanced for milk production. Research was conducted on
Farm 2 (n = 360) between May and June 2002. Farm 2 was located in southwestern
Kansas (7000 lactating cows) and had a rolling herd average of 9798 kg. Cows were

milked twice or thrice daily depending on production and DIM. Cows were fed primarily

32

corn silage, alfalfa hay and corn diet balanced for milk production. Research was
conducted at Farm 3 (n=1 1 10) between October 2002 and April 2003. Farm 3 was
located in southern Michigan (2500 lactating cows) and had a rolling herd average of
10,886 kg. Cows were milked three times daily and were fed a corn silage and haylage
based ration balanced for milk production.

Cows received the following estrous synchronization protocol: Pre Synch (two

injections of PGFZa 14 days apart) followed on d 14 by the Ovsynch program. The

Ovsynch program used in this study consisted of an intramuscular injection of 100 p g
gonadotropin releasing hormone (GnRH; Cystorelin ®, Merial Ltd., Duluth, GI.)

followed in 7d with 25 mg PGFZa (Lutalyse ®; Pharmacia-Upjohn Co., Kalamazoo, MI)

then, 40-48 h later, with 100 u g of GnRH (Figure 1). Insemination took place at either -8
or 16 h after the second injection of GnRH. Farm personnel administered all injections

except the ﬁnal GnRH, which investigators administered to assure precision and timing.

GnRH PGFZa AI -8 h GnRH AI 16 h
(100p g ) (25 mg) (Treatments 1 & 3) (100p g ) (Treatments 2 & 4)
l 7 d l 36 h I 8 h l 16 h I
L I I I _I

 

Figure 2. The timing for each injection used in the Ovsynch protocol and timing of Al
for all cows on study.

One AI technician performed insemination for all cows, either within the uterine
body or deep in the uterine horn ipsilateral to the ovary with the ovulatory follicle. The
technician was trained for unilateral horn insemination. Beal et al. (1991) established the

training protocol we used. The AI technician used an insemination gun with a metal

33

swivel attached to the end. The technician deposited the metal swivel into the uterine
horn. With ultrasonography, proper placement was veriﬁed. This technique was
effective in determining competency of the inseminator for proper placement of semen
deep in the uterine horn. Proper uterine horn deposition was achieved when resistance
was felt after slight straightening or manipulation of the horn.

Only cows responding to Ovsynch with a synchronized ovulation were included
in the study. An Aloka 500V ultrasound machine with a 7.5 MHz linear array transducer
probe (Corometrics Medical Systems Inc., Wallingford, CT) was used to evaluate ovaries
and diagnose pregnancy. Ovarian structures were mapped using ultrasound immediately
before insemination to determine size of the ovulatory follicle and presence of corpora
lutea (CL). This information was used to establish which horn would be ipsilateral to the
ovulatory follicle. Once the horn ipsilateral to the ovulatory follicle was determined, one
straw of semen was placed either in the ipsilateral horn or uterine body depending on
treatment. Two days after AI, cows were re-examined by ultrasound to determine a
synchronized ovulation. A cow was considered synchronized if the ovulatory follicles
(from the previous scan) had disappeared or a new CL was present. The scan was also
used to conﬁrm proper semen placement in the horn ipsilateral to the ovulatory follicle
for cows inseminated in the uterine horn. Pregnancy diagnoses were determined between
28 to 33 d (hereafter referred to as 28 d) post- insemination for all treatment groups.
Observation of a fetal heartbeat was required before pregnancy was conﬁrmed and
recorded. A second diagnosis was performed at 56 to 60 d (hereafter referred to as d 56)
post insemination for cows previously conﬁrmed pregnant at d 28.

The total number of cows inseminated in this study was 2347, but only 1603 cows

34

were included in the analyses. The synchronization rate for all cows was approximately
83%. Cows were excluded (n = 744) from the analysis if ovulation was not observed, if
cows were bred < 5 days following experimental insemination, or the AI technician could
not deposit semen in the designated location because of an impassible tract. Cows culled
before both pregnancy diagnoses were also excluded.

Service sires for Farm 1 and Farm 2 were selected by farm management.
However, on Farm 3 service sires were selected based on ERCR values and evaluated for
fertility. Three bulls were selected that had +4 ERCR and two with -4. All bulls had a
repeatability > 95 % from Select Sires, American Breeders Service (ABS), or CRI Genex
Cooperative, Inc. Semen from each of the ﬁve sires was evaluated for concentration and
motility post-thaw. We randomly selected 5 straws (0.05 ml) of semen to be evaluated
from each of the bulls. Semen was diluted (1: 10) with modiﬁed Tyrode’s (mTALP)
media to maintain motility (Liu and Foote, 1998). Immediately after semen was diluted,
a drop from the semen sample was placed on a slide and covered with a cover slip. The
percentage of sperm moving progressively forward was estimated at 400x under a
microscope. Concentration of sperm was determined by haemocytometry.

Semen from the ﬁve service sires were randomly allocated to cows in each of the
four treatment groups by parity and days in milk. AI technician and ultrasound operator
at pregnancy diagnosis were blind to bull selection and treatment.

Management factors that could affect fertility in the lactating dairy cows were
also evaluated. These factors included lactation information, uterine health information,
bST status, SCC near the time of AI, parturition data, BCS, and locomotion scores. For

all farms, cows that failed to expel placental membranes within 24 h after calving were

35

diagnosed as a RP. Each of the three farms had a different regime for bST
administration. Farm 1 started cows on bST at 63 DIM if cows exhibited a BCS of at
least 2.3. Farm 2 started cows on bST 70 DIM and Farm 3 started cows at 65 DIM.
Cows were either recorded as receiving bST at the time of insemination or not receiving
bST; actual injection dates of bST were not recorded. Approximately 94 % of the cows
on the study received bST by the time of AI.

Researchers scored cows at the time of the second ultrasound observation (2 (1
following inseminations) for both locomotion and body condition. Locomotion scores
were based on a 1 to 5 scale adapted from Sprecher et al. (1996). A score of 1 is a
normal cow that stands with a level top line and walks with a level top line. A score of 2
is a mildly lame cow that stands with a level top line but walks with an arched back. A
score of 3 is a moderately lame cow that stands with an arched back and walks with an
arched back. A score of 4 is a lame cow that stands and walks} with an arched back and
has a noticeable limp. A score of 5 is a severely lame cow that has an arched back, does
not bear any weight on one leg and may have difﬁculty getting up from the lying down
position.

BCS was evaluated on a ﬁve point scale modiﬁed from (Wildman et al., 1982). A
cow with a score of 1 is emaciated with vertebrae prominent and hooks and pin bones
sharply deﬁned. The thurl region and thighs are sunken and in curving. The tail head
depression is severely receded and the vulva appears prominent. A cow with a BCS
score of 2 is thin, with less prominent vertebrae. The hook and pin bones are prominent
but the depression of the thurl region between them is less severe than a cow with a BCS

of 1. The area around the tail head is also less sunken and the vulva less prominent. A

36

cow with a BCS score of 3 is in average body condition. The backbone is a rounded ridge
and hook and pin bones are round and smoothed over. The tail head area is ﬁlled out but
there is no evidence of fat deposits. A cow with a score of 4 is in heavy condition. The
ridge of the backbone is ﬂattening over the loin and rump areas and rounded over the
chine. The hook bones are smoothed over and the span between the hook bones over the
backbone is ﬂat. The area around the pin bones shows patches of fat deposits. A cow
with a BCS score of 5 is extremely heavy conditioned. The bone structure of the top line,
hook and pin bones and the short ribs are not visible. Fat deposits around the tailbone
and over the ribs are obvious. The thighs curve out; the brisket and ﬂanks are heavy and
the chine very round.

Cow information such as parity, number of AI services (all services for current
lactation, including experimental insemination), milking frequency, milk yield (nearest
the time of insemination), SCC nearest the time of insemination (SCC and milk
measurements taken every 2 weeks) and whether cows had received bST around at the
time of breeding were retrieved from farm computer records. Somatic cells were
recorded as a count on two of the farms record systems. Counts were converted to
somatic cell score (SCS) using the following formula: [logz (SCC/ 100,000) +3]. Cows
with previous calving information such as RP or twins were recorded. Additionally,
calving difﬁculty score (based on PCDART Protocol 1-5) for the previous calving was

collected.

Statistical Analyses

Independent analyses were considered for the three response variables (pregnancy

37

diagnosis at 28 d, pregnancy diagnosis at 56 d, and pregnancy 1033), using a generalized
linear model technique (McCullagh and Nelder, 1989). The analyses were performed
using the procedure GENMOD of the SAS System (SAS, 1999), with a binomial
distribution and the canonical link function. Pregnancy rate per AI (PR/AI) is the number
of cows diagnosed pregnant divided by the total number of cows Al. The difference in
pregnancy rate between 28 and 56 (1 post insemination provided early pregnancy 1053
information. Preliminary analyses were performed including each management factor
separately in addition to treatment effects (time, site of insemination, time x site of
insemination). The ﬁnal analysis started with a ﬁrll model including all factors and ﬁrst
order interactions. Non-signiﬁcant interactions were then removed from the model.
Continuous data such as milk production, DIM, follicle size, CL size, and SCS were
grouped into classes based on either a statistical distribution or biological factors
established from known information or previous research practices. Some analyses were
performed on selected cows. The effect of follicle size at the time of insemination on
pregnancy outcome is one example of selection. We used only single ovulating cows
because cows with multiple ovulations were found to have an advantage in fertility and
would have introduced bias. When evaluating milk production and parity, we selected
only ﬁrst service cows 60 to 90 DIM. This was done to form a consistent set of cows to
test the actual effect of milk production and parity on cows at the same stage of lactation
with the same reproductive status. Finally, using a similar approach, the ﬁve selected
sires were analyzed for PR/AI differences for time and site of insemination in cows at
Farm 3. Tables were constructed for service sire x site and time of insemination to

illustrate additional fertility information for each of the ﬁve sires. In addition to the ﬁeld

38

fertility data, we also compared 5 straws of semen from each of the ﬁve bulls using the
non-parametric ANOVA technique of Kruskal-Wallis (Zar, 1999), for concentration and
motility differences.

A preliminary analysis of the effect of timing of Al on gender ratio was
performed by comparing percentage of female calves using the chi-square goodness-of-
ﬁt test to an expected population value of 45.8% females and 54.2 % males (Ryan and

Boland, 1991). A ﬁnal analysis will be completed once all calves for this study are born.

39

RESULTS

PR/Al for cows inseminated at all farms and all treatments combined at 28 and 56
d were 34.3 and 28.0 %, respectively (Table 2). The percent of embryo survival between
28 and 56 d after insemination was 81.9%. Interactions of farm x time of insemination
and farm x site of insemination were not signiﬁcant. Therefore, to evaluate the effect of
farm, all treatments were combined. There was however, an effect of farm, with Farm 1
having the highest fertility (P = 0.04) at 28 d post insemination across treatments.

Pregnancy rate following insemination 8 h before or 16 h after the ﬁnal injection
of GnRH of the Ovsynch with semen deposition occurring in either the uterine body deep
uterine horn is depicted in Table 3. The 16 h treatment groups had a greater percentage
of cows pregnant than the -8 group at 28 d (P < 0.01) and 56 d (P < 0.01) following AI.
There was less pregnancy loss in cows inseminated in the uterine body (P < 0.05) than
the uterine horn.

Compared with an expected gender ratio of 46: 54 (female: male) ratio based on
(Ryan and Boland, 1991) there were more (P = 0.01) female than male calves born after
A1 at -8 h than would be expected (Table 4). Gender ratio in the 16 h group did not
signiﬁcantly differ when compared with the expected ratio.

At Farm 3 there was a signiﬁcant difference in fertility among the ﬁve service
sires at 28 and 56 d (P < 0.01) following A1 at -8 or 16 h (Table 5). However, there was
no interaction of sire x time of insemination. Table 6 shows the difference in fertility or
service sires based on site of insemination. Again, the percent cows pregnant at diagnosis

differed among sires (P < 0.01). No interaction of sire x site of insemination for the ﬁve

40

sires at 28 and 56 d following Al was found. Finally, a comparison post-thaw semen
analysis is depicted in Table 7. It appears that the 1 and 5 bulls have the lowest motility
and the highest live concentration of sperm per straw. There was not signiﬁcant variation
between the ﬁve straws of semen evaluated for each bull.

PR/AI by parity for ﬁrst service cows 60 to 90 DIM for either above or below
mean milk production (40.4 kg/d) is depicted in Table 8. There was a trend for cows
with milk production greater than the mean to have higher fertility at 28 (P = 0.07) and
56 d (P = 0.09) following AI than cows with below average milk production. The effect
of parity on pregnancy rate at 28 and 56 d is depicted in Table 9. Parity for all cows
regardless of stage of lactation or number of services had no signiﬁcant difference in
PR/AI at 28 or 56 (1 following AI.

PR/AI for single ovulating cows with follicles larger than 12 mm at the time of AI
were greater at 28 d (P = 0.01) and 56 d (P = 0.02) compared to cows with < 12 mm
follicles (Table 10). The effect of multiple ovulations per cow on PR/AI at 28 and 56 d is
depicted in Table 11. PR/AI for cows with multiple ovulations were greater at 56 (1
following AI (P = 0.02) than single ovulating cows. There was a trend for greater
fertility in multiple ovulating cows at 28 (I post insemination (P = 0.07) and less
embryonic 1035 (P = 0.08) than for single ovulating cows.

Table 12 depicts the relationship between CL size at the time of insemination and
28 d and 56 d PR/AI and pregnancy loss. Larger CL ( 3 17 mm) at the time of
insemination decreased the percent of cows pregnant at 56 d and increased pregnancy
1033 (Table 12). Additionally, PR/AI in cows with 3+ locomotion score were greater at

28 (1 following uterine body (P = 0.05) than uterine horn Al (Table 13).

41

PR/AI for all treatments combined by number of AI services for current lactation
is depicted in Table 14. There was a clear trend for ﬁrst service cows to have higher
PR/AI than cows with multiple services, but data were not signiﬁcant at (P < 0.05).

SCS near the time of A1 did not affect the percent of cows pregnant at 28 or 56 (1
following AI (Table 15). There were no interactions for SCS x time or site of
insemination. In addition, PR/AI and pregnancy 1053 between 28 d and 56 d was not
different for cows in different stages of lactation (Table 17).

There was no effect of calving difﬁculty score from the previous calving on
PIUAI (Table 16). Cows with or without prior calving difﬁculty had similar fertility.
Additionally, the effect of BCS on percent pregnancy for all treatments combined is
presented in Table 18. Cows with > 3.46 BCS tended to have higher pregnancy rate at

56 d and less pregnancy 1033 then cows with lower BCS.

42

Table 2. The effect of farm on percent of lactating dairy cows pregnant at 28
and 56 d following A1. a

 

 

 

 

Farm 1 Farm 2 . Farm 3 SRO/til P-value b
n 133 360 1110
28 d 44.1 33.8 33.5 34.3 0.04
56 d 34.8 26.4 27.9 28.0 0.11
Loss c 20.3 22.1 16.3 18.1 0.22

 

a There were no interactions for farm x site of insemination or farm x time,
therefore all treatments were combined.

b P-value indicates differences between farms

° Percent pregnancy 1055 between 28 and 56 d.

43

Table 3. Effect of timing of AI relative to the ﬁnal GnRH injection of
Ovsynch and site of insemination on percent lactating dairy cows pregnant at
28 and 56 d.

 

Time of Site of a

 

 

 

 

 

insemination insemination n 28 d 56 d 1053
Body 400 30.4 25.7 15.6
-8 h Hom 372 28.6 21.5 24.5
Total PR/AI -8 h 29.3 * 23.4 * 20.0
Body 417 40.4 34.3 14.9
16 h Horn 414 37.7 30.4 18.8
Total PR/AI 16 h 39.0 32.3 16.8
Total PR/AIfor body 35.4 29.9 15.3 **
Total PR/AI for horn 33.2 26.0 21.3

 

3 Percent pregnancy 1053 between 28 and 56 d.

*Iess than value at 16 h (P < 0.01)
“Less than value for horn insemination (P < 0.05)

44

Table 4. Percent female and male offspring born to lactating dairy cows at

Farm 1 and 2, inseminated at -8 h or 16 h relative to ﬁnal GnRH injection of
Ovsynch.

 

 

 

 

Gender Ratio
Time of . a
Insemination Female . Male n P-value
-8 h 65.2 :34.8 46 0.01
16 h 56.1 :43.9 66 0.09
n 67 45 112

 

a P-value indicates difference between -8 and 16 h to the expected female: rmle
gender ratio (45.8: 54.2) obtained from Ryan and Boland (1991).

b There was no effect of site of insemenation on gender ratio, therefore all
treatments were combined. '

45

Table 5. Effect of service sire and timing of AI relative to ﬁnal GnRH
injection of Ovsynch on percent lactating dairy cows pregnant at 28 and 56 (1
following A1 at Farm 3.

 

 

 

 

 

 

 

28 d 56 (1 Loss 8
Sire ERCR -8 (n) 16 (11) -8 (n) 16 (n) -8 16
1 -4 17.7 (68) 29.6 (98) 10.3 (68) 24.7 (97) 41.7 14.3
2 .4 34.1 (88) 46.8 (94) 27.3 (88) 42.6 (94) 20.0 9.1
3 +4 45.3 (95) 44.0 (116) 41.1 (95) 43.1 (116) 9.3 2.0
4 +4 35.4 (82) 38.5 (91) 26.8 (82) 31.9 (91) 24.1 17.1
5 +4 24.1 (87) 33.0 (88) 20.7 (87) 28.7 (87) 14.3 10.7

Name b <0.01 <0.01 0.01

Interaction ° 0.44 0.33 0.66

P—value

 

3 Percent of pregnancy 1033 between 28 and 56 d
b P-value for difference among sires within -8 and 16 h and for the average of the two

C P-value for the interaction of sire x time of insemination

46

Table 6. Effects of service sire and site of semen deposition, either uterine
body or deep uterine horn, on percent lactating dairy cows pregnant at 28
and 56 (1 following Al on farm 3.

 

 

 

 

 

 

28 d 56 (1 Loss "
Sire ERCR Body (11) Horn (11) Body (11) Horn (11) Body Horn
1 -4 26.8 (82) 22.6 (84) 22.2 (81) 15.5 (84) 14.3 31.6
2 -4 38.8 (85) 42.3 (97) 34.1 (85) 36.1 (97) 12.1 14.6
3 +4 42.5 (106) 46.7 (105) 40.6 (106) 43.8 (105) 4.4 6.1
4 +4 39.6 (91) 34.2 (82) 30.8 (91) 28.1 (82) 22.2 17.9
5 +4 26.7 (86) 30.3 (89) 25.6 (86) 23.9 (88) 4.4 19.2
P-value b <0.01 <0.01 0.01
Interaction P-value c 0.78 0.76 0.36

 

a Percent pregnancy 1033 between 28 and 56 d
b P-value for difference among sires within -8 and 16 h and for the average of the two

c P-value for the interaction of sire x time of insemination

47

Table 7. Semen evaluation (for concentration and motility) for each of the
ﬁve bulls used to AI cows at Farm 3.

 

 

 

Bull a ERCR Live :plearvrvn per :leorfﬂeip; Total live 1:10;]; sperm per
1 - 4 7,450,000 :I: 539,965 0.49 i 0.01 3,633,750 :t 207,379
2 - 4 4,000,000 i 410,791 0.75 i 0.02 3,000,000 a: 316,653
3 + 4 4,525,000 i 632,949 0.67 i 0.03 2,978,750 3: 344,791
4 + 4 4,150,000 :t 449,652 0.69 i 0.02 2,840,000 i 265,066
5 + 4 8,825,000 i 979,756 0.56 i 0.02 4,883,125 :t 421,515
P-value < 0.01 < 0.01 0.02

 

a Each estimate for semen evaluated per bull was based on (n = 5) randomly selected 0.05
cc. straws of semen

48

Table 8. Effect of milk production (above or below the mean) for lst service
dairy cows 60-90 (1 in lactation on percent pregnant at 28 and 56 (1 following
A1. a

 

Milk Production (Mean=40.4 kg)

 

 

 

 

 

 

Above Below Above Below Above Below
Parity 28 d (n) 56 d (11) Loss b
1 31.8 (151) 35.4 (178) 26.7 (150) 28.7 (178) 14.9 19.1
2 41.3 (63) 26.1 (46) 31.8 (63) 22.2 (45) 23.1 9.1
3+ 51.1 (92) 39.7 (73) 45.1 (91) 31.5 (73) 10.9 20.7
P-value 0.07 0.09 0.99

 

3 There were no interactions for milk production and parity by treatment eﬁects, so all
treatment groups were combined.

b Percent pregnancy 1033 between 28 and 56 d

49

Table 9. Effect of parity on percent lactating dairy cows pregnant at 28 and
56 (1 following AI.

 

 

 

 

Parity
l 2 3+ P-value
28 d 34.2 31.8 36.9 0.30
56 d 28.6 24.4 29.7 0.26
Loss 3 16.4 22.8 18.8 0.42
n 916 321 366

 

3 Percent pregmncy 1053 between 28 and 56 d

50

Table 10. Effect of follicle size at time of insemination on percent single
ovulating lactating dairy cows pregnant at 28 and 56 d following A1. a

 

Follicle size (mm)

 

 

_<_12 >12 P-value
28 d 27.2 35.6 0.01
56 d 21.5 28.7 0.02
Loss 3 20.9 18.7 0.90

 

 

 

n 480 944

 

3 Percent pregnancy 1033 between 28 and 56 d

51

Table 11. Effect of multiple ovulations on percent lactating dairy cows
pregnant at 28 and 56 d following A1. a

 

 

Multiple b
Ovulations n 28 d 56 d Loss
Yes 179 40.4 36.1 10.8
No 1424 33.7 27.1 19.1

 

P-value 0.07 0.02 0.08

 

aThere were no interactions for multiple ovuhtions x time or site of
inseminantion, so all treatments were combined.

b Percent pregnancy 1033 between 28 and 56 d.

52

Table 12. Effect of corpus luteum (CL) size at time of insemination on
percent lactating dairy cows pregnant at 28 and 56 (1 following AI.a

 

 

 

CL Size (m)
< 11 11-139 14-159 16-16.9 > 17 P-value
28 d 35.7 31.8 36.1 31.9 22.5 0.13
56 d 29.6 27.3 29.7 23.5 12.4 0.01
1033 b 17.1 12.8 16.7 23.8 45.0 0.05
n~ “1175— I —151 T _11; I '69” T89— - T — —

 

" There was no interaction for CL size x time or site of insemination, therefore all
treatments were combined.

bPercent pregnancy 1033 between 28 and 56 d.

53

Table 13. Percent lactating dairy cows pregnant at 28 and 56 d following AI
by site of insemination and scored for locomotion. a

 

Locomotion Score

 

 

 

 

. Site of Interaction
Vanable insemination 1 (n) 2 (11) 3+ (n) P-value
Body 34.3 (560) 33.6 (155) 44.1 (84)
28 d 0.05
Horn 33.0 (533) 35.8 (162) 26.7 (75)
Body 29.3 27.1 36.2
56 d 0.14
25.9 26.9 22.7
Horn
Body 14.6 19.2 16.7
1035 b 0.69
21.1 23.2 15.0
Horn

 

3 There was no interaction for time x locomotion score (p > 0.05)

b Percent pregnancy 1033 between 28 and 56 d

54

Table 14. Effect of number of services on percent lactating dairy cows

pregnant at 28 and 56 d following AI.at

 

Number ofAI services

 

 

 

1 2 3 3 P-value
28 d 37.2 31 7 32 6 0.08
56 d 30.6 25 4 26 5 0.09
L053 b 17.0 19 8 18 7 0.76
n TEETTEETTEZT—‘T—m—

 

3 There were no interactions of AI services x time or site of insemination,

so all treatments were combined.

b Percent pregnancy 1033 between 28 and 56 (1.

**AI Services include experimental insemination in addition to previous

services within current hctation.

55

Table 15. Effect of somatic cell score (SCS) near the time of insemination
on percent lactating dairy cows pregnant at 28 and 56 d following AI.a b

 

 

 

 

SCS
< .6 .6 - 1.7 1.7 - 3.3 > 3.3 P-value
28 d 34.7 32.9 32.1 37.5 0.38
56 d 27.8 27.7 26.9 29.6 0.86
1035 C 19.3 15.8 16.3 20.7 0.59
n 409 389 402 403

 

a The somatic cell score was recorded based on the test day that was nearest
day of insemination.

b There were no interactions for SCS x time or site of insemination (P < 0.05),
therefore all treatments were combined.

c Percent pregnancy 1033 between 28 and 56 d.

56

Table 16. Effect of previous calving difﬁculty score on percent lactating

dairy cows pregnant at 28 and 56 d following A1. a

 

Calving Difﬁculty Score

 

 

 

0 - l > 1 P-value
28 d 36.4 38.4 0.76
56 d 28.2 31.3 0.59
1033 a 22.4 18.4 0.48
n 396 95

 

3 Percent pregnany 1035 between 28 and 56 d.

57

Table 17. Effect of days in milk (DIM) on percent lactating dairy cows

pregnant at 28 and 56 d following A1. a

 

 

 

 

 

 

DIM
< 75 75 - 150 > 150 P-value
28 d 35.8 34.4 33.5 0.63
56 d 29.1 28.4 26.7 0.54
1035 b 18.6 17.0 20.0 0.66
n T 3;— - 912 375 #—

 

 

 

3 There were no interactions for DIM x time or site of insemination, therefore treatments

were combined.

b Percent pregnancy 1033 between 28 and 56 d

58

Table 18. Effect of body condition score (BCS) on percent lactating dairy
cows pregnant at 28 and 56 (1 following A1. a

 

 

 

 

 

 

BCS
1.0 - 2.35 2.36 - 3.45 3.46 - 4.0 > 4.0 P-value
28 d 30.3 33.2 37.2 31.3 0.25
56 d 23.2 25.7 31.5 28.1 0.07
Loss 3 21.9 22.2 15.1 10.0 0.08
:1— I I 1.109 TT 754 I. 592 A 128—— ———-

 

3 Percent pregnancy 1053 between 28 and 56 d

59

DISCUSSION

This study was designed to determine the effect of prolonged subsistence of
sperm in the female reproductive tract prior to ovulation in lactating dairy cows, with two
different sites of Al, on PR/AI and resulting gender ratio. Precise synchronization of
ovulation (Ovsynch) allowed for the opportunity to test timing of AI in relation to
ovulation. In a previous study (Pursley et al., 1995), Ovsynch synchronized ovulation 24
to 32 h following the ﬁnal GnRH induced LH surge. Previous studied demonstrated that
A1 at the time of the ﬁnal GnRH of Ovsynch (~ 28 h prior to ovulation) decreased the
PR/AI but increased the number of female calves born. In the current study time of AI
prior to ovulation to ~ 36 h (-8 group) was extended to determine if female to male ratio
could be improved from previous work and if fertility could be maintained by deep
uterine horn AI. Controls received AI 16 h following the GnRH induced LH surge (16 h
group; ~ 12 h prior to ovulation). This time was previously proven to be the ideal time of
AI prior to ovulation (Pursley et al., 1998). Since there was no effect of site of
deposition, we will discuss the effects of timing of A1 with the two sites of AI combined

within each group.

Effect of Extending Sperm Subsistence in the Uterus / Oviduct Prior to Ovulation
Our results indicated that the female: male ratio was substantially shifted in favor

of females born from cows in the -8 h group. Our results, 65 % females, differed from

the expected value of 46 % females reported by Ryan and Boland (1991). These results

are consistent with data from Pursley et al. (1998) that reported more heifers when AI

60

occurred ~28 h prior to ovulation. There are phenotypic differences between X- and Y-
bearing sperm including plasma membrane surface charge, density, morphology (nucleus
and head), and motility, and the degree of motility measured in effective velocity was
greater in X-bearing sperm than in Y-bearing sperm (Johnson, 1995; Windsor et al.,
1993). These differences may allow the X-chromosome-bearing sperm to have greater
longevity than the Y-chromosome-bearing sperm. Thus, extending the time sperm are
present in the uterus before ovulation may result in selected death of the Y-chromosome
bearing sperm. However, it appears that the potential deviation in X vs. Y sperm takes a
minimum amount of time. Prolonged pre-incubation of sperm for 20 h prior to in vitro
fertilization increased the number of female hatched blastocysts (Lechniak et al., 2003).
Substantial differences in sex ratio with regard to the relationship of time of insemination
or intercourse and ovulation have been reported in other species (Guerrero, 1974; Hart
and Moody, 1949; Hedricks and McClintock, 1990; Pratt et al., 1987; Verme and Ozoga,
1981). From these studies along with the current study, it appears that insemination
around 2 l (1 prior to ovulation results in more female offspring compared with a
preponderance of males when insemination occurs around the time of ovulation. Some
previous studies in cattle (Ballinger, 1970; F oote, 1977; France et al., 1984; Rorie et al.,
1999) indicated that there is no relationship between time of AI and sex ratio. However,
the design of the current study allowed for a more accurate prediction of time of
ovulation, therefore extending the time that sperm were forced to subsist from A1 to
ovulation. The current study also had greater numbers of births to evaluate gender ratio

in comparison to previous studies in cattle.

61

It is not clear from results to date if semen from bulls with a signiﬁcant difference
in fertility will perform similarly with regard to female: male ratio when inseminated into
cows at various times from ovulation. Calving data from the 5 bulls on Farm 3 will be
analyzed separately and compared to the expected gender ratio for dairy cows once all
calves are born.

When sperm resided in the reproductive tract for ~36 h before fertilization, there
was only a 9 % decrease in PR/AI at 56 d post-AI compared to controls. This decrease
in PR/AI was not as dramatic as expected, based on previous ﬁndings that sperm may
only survive in the female reproductive tract for approximately 30 h (Laing 1945).
However, more recent studies suggested that frozen/thawed sperm may live up to 48 h
following AI (Bazer et al., 1987). It is not clear if fertilization rates were similar between
-8 and 16 groups and more embryonic losses occurred from fertilization to the 28 (1
pregnancy diagnosis, or if the difference in fertility was due to differences in fertilization
rates. However, when Lechniak et al. (2003) incubated sperm for 20 h prior to in vitro
fertilization there were reduced development and cleavage rates compared to no pre-
incubation.

Pregnancy loss was similar between the -8 h and 16 h groups. Pursley et
al.(1998) found fewer pregnancy losses when cows were inseminated at the time of
GnRH injection. The 18 % pregnancy 1033 between 28 and 56 d post-AI found in this
study appears to be similar to other studies (Pursley et al., 1998; Smith and Stevenson,
1995) when ultrasound was used to detect pregnancy at 28 d after A1. Vasconcelos et al.
(1997) found an 1 l % 1035 between 28 and 42 d and an additional 6 % 1053 between 42

and 56 d after AI.

62

It is well documented that service sires differ in their ability to fertilize oocytes in
vitro (Marquant-Le Guienne et al., 1990; Ward et al., 2001b). Highly fertile beef bulls
can be identiﬁed by the fertility associated antigen (FAA). Approximately, 60 % of beef
bulls screened tested positive for this antigen. In a ﬁeld study by Sprott et al. (2000),
bulls that tested positive for FAA had increased PIUAI compared to bulls that tested
negative for the antigen (68 vs. 47 %). However, nearly all dairy bulls thus far screened
have tested positive for this antigen (Dr. Roy Ax, personal communication). To our
knowledge, the current trial is the ﬁrst in vivo study that compares service sire fertility
following prolonged subsistence of sperm in the female reproductive tract for ~ 36 h prior
to ovulation. We used the ERCR as a guide to select high and low fertility bulls. The
ERCR is based on 70 d non-retum rate, which is currently the best estimate of fertility
available for selection of dairy bulls. However, it is not clear how accurately ERCR
accounts for fertility in dairy bulls. Therefore, we did not base our analysis on the ERCR
but rather on the individual bull.

The two insemination times chosen for the current study (-8 and 16 h) provide a
novel way to evaluate bull fertility when sperm are inseminated in the female
reproductive tract for an extended period of time prior to ovulation. Bull 3, the bull with
the greatest PR/AI for this trial, had similar PR/AI at -8 compared to the 16 h treatment.
In addition, this bull had fewer pregnancy losses between 28 and 56 d following AI. In
contrast, Bull 1 had the lowest PR/AI, the greatest abatement in PR/AI in the -8 group,
and the greatest pregnancy 1035 between 28 and 56 d post-Al. Other investigators
(Callaghan and King, 1980; Nadir et al., 1992) also demonstrated a signiﬁcant deviation

in high vs. low fertility bulls. Ward et al. (2001a) studied the effect of bull fertility on the

63

kinetics of early embryonic development and found that embryos sired by high fertility
bulls had a faster rate of early embryonic development.

The difference in performance amongst bulls in our study is probably not
attributed to concentrations of live motile sperm inseminated. Numbers of live motile
sperm were similar amongst Bulls 2, 3 and 4, and greater in Bull 1 and 5. The only
sperm characteristic we evaluated that appeared to be associated with fertility was percent
motility. The two bulls with the lowest motility, Bull l and 5, also were the lowest
fertility bulls.

Perhaps the most interesting aspect of the bull fertility data is the effect of bull on
pregnancy 1033 between 28 and 56 d post-AI. It was clear that the highest fertility bull
had the fewest pregnancy losses and the lowest fertility bull had the greatest losses. This
is the ﬁrst study that we are aware of that associates pregnancy loss subsequent to A1 to
the fertility outcomes of the service sire. From these data it appears that pregnancy loss

may be partially predetermined at the time of fertilization.

Effect of Site of Semen Deposition on Fertility

It appears that improving percentage of female offspring by timing AI ~36 h
before ovulation may come with a price. We hypothesized that deep uterine horn
insemination would maintain PR/Al in lactating dairy cows inseminated in the -8 h group
compared to the 16 hour group. However, there was no difference in PR/AI in uterine
body vs. deep uterine horn inseminated cows. In four unpublished studies from our lab,
we have detected a signiﬁcant increase in fertility using deep uterine horn AI. In two of

those studies, we detected an interaction of site of AI and AI technician. Unfortunately,

64

differences in Al techniques between these technicians have not been identiﬁed. The
current study used one AI technician for all cows. This technician was trained for
unilateral horn insemination prior to the study using a novel training technique that Beal
et al. (1991) established using ultrasound. To minimize variability and strengthen our
analysis we decided to use only one AI technician. This was an advantage when
evaluating sire fertility. One technician reduces the variability, allowing the focus to be
on the effect of the sire. However, a disadvantage is that we were unable to determine
whether inseminator technique affected fertility based on site of semen deposition.

The proposed advantage of uterine horn insemination (bicomual or unicomual)
when compared with body insemination is a greater likelihood of deposition of semen
nearer the uterotubal junction and reduced incidence of cervical deposition (Lopez-
Gatius, 2000). Many investigators have studied bicomual insemination, depositing half
the semen in one uterine horn and half in the contralateral hem. However, varying
results have been noted (Gallagher and Senger, 1989; Graves et al., 1991; Knight et al.,
1951; McKenna et al., 1990; Senger et al., 1988; Williams et al., 1988). Senger et al.
(1988) showed a dramatic increase in CR using deep bicomual insemination. CR were
improved from 45% to 70% in 4000 cows. In the current study we used unicomual
insemination. Similar to Zavos et al. (1985), we administered a full dose of semen deep
into the uterine horn ipsilateral to the side of impending ovulation. Uterine horn
insemination did not increase fertility when compared to uterine body insemination. This
is in conﬂict with other unilateral insemination data previously reported (Lopez-Gatius,
1996; Lopez-Gatius and Camon~Urge1, 1988; Zavos et al., 1985) that showed marked

improvement in fertility. Additionally, we observed signiﬁcantly higher pregnancy loss

65

between 28 and 56 (1 post insemination for cows inseminated unilaterally in the uterine

horn.

The Effect of Other Physiological and Management Factors on Fertility

Reproductive performance is an intricate part of a successful dairy management
program. Reproductive performance is determined by the relationship of management,
environment and physiological factors. In this study we also examined the relationship
between some speciﬁc physiological and management factors and reproductive
performance.

Parity is a physiological factor that affects fertility. Hillers et al. (1984) found
that cows in third and greater lactations had lower CR and longer intervals to ﬁrst service
than ﬁrst and second lactation cows. However, we did not ﬁnd a difference in fertility
among 1, 2 or >3 parity cows.

Multiple ovulations are also associated with parity and high production of milk in
lactating dairy cows (Fricke and Wiltbank, 1999). However, our results did not indicate
a higher likelihood of multiple ovulations in high producing or older cows. We did
discover that cows with multiple ovulations (12.6 % of all cows) had a higher pregnancy
rate at 56 d with a trend for less pregnancy 1033. Although we do not have information on
number of CL at 56 d post AI, multiple CL present on the ovary may be an explanation
for lower pregnancy loss. Lopez-Gatius et al. (2002) found that cows carrying single
calves with two CL were less likely to lose the pregnancy.

Along with parity, we also evaluated the relationship between stage of stage of

lactation and fertility. Previous studies reported a higher risk of pregnancy failure for

66

cows that conceived in early and in late lactation (Loefﬂer et al., 1999). However, in the
current study with a large sample size, we saw no difference in PR/AI for cows in
different stages of lactation.

Suckled beef cows with follicles > 12 mm on d 2 following insemination had
greater PR than those with follicles 5 12 mm (Lamb et al., 2001). Our results indicate a
similar relationship between follicle size and fertility. Single ovulating cows with
follicles > 12 mm at the time of insemination had a greater PR/AI at 28 and 56 (1 post
insemination than cows with 5 12 mm follicles. A by-product of the Ovsynch program
may be the synchronization and ovulation of smaller less competent oocytes. This may
explain why cows with < 20 mm follicles have reduced fertility.

We are unaware of any data on CL size at the time of insemination and PR/AI and
pregnancy loss in lactating dairy cows. Cows in current trial with a CL > 17 mm at the
time of insemination had the lowest PR/AI. Perhaps, low fertility in cows with a large
CL may be an indication of poor synchronization. Unfortunately, we did not collect CL
regression information. Nonetheless, it is difﬁcult to understand why some cows with a
large CL at insemination conceived but consequently had an increased chance of
pregnancy loss. The cause of this is unclear, but it may potentially be attributed to
hormone levels at the time of fertilization and subsequent effects on development.

There was also a trend for ﬁrst AI service cows to have a slightly higher percent
pregnancy at 28 and 56 (1 post insemination. This could be attributed to cows that have
fewer reproductive disorders or responded better to the Ovsynch program.

Novel information on potential management factor effects on fertility can be

gained from the current study. One management factor that could directly affect fertility

67

is cow lameness. Locomotion scoring provides producers with a way to assess the
amount of lameness in their herd. Previous studies indicate lameness can have a
detrimental affect on fertility (Hernandez et al., 2001; Melendez etal., 2003). Our data
indicated an unexpected interaction between locomotion score x site of insemination.
Cows with >3 locomotion scores inseminated in the uterine horn had the lowest fertility.
However, over interpretation of these data is discouraged because cows with a three
locomotion score were not severely lame. Cows with a locomotion score of 3 may be
predisposed to lameness in the ﬁiture or recovering from an injury. However, a true
evaluation of effects of severe lameness on fertility would require more animals with >4
locomotion score.

BCS is another management tool that producers can use to better manage cows.
BC S can be used to adjust dairy herd rations and herd health programs to achieve the
most cow productivity, fertility and longevity. Butler and Smith (1989) reported that, on
average, cows resumed estrous cyclicity 10 (1 following the nadir in energy balance. One
mechanism controlling estrous cyclicity in cows is LH pulsatility. LH secretion is
suppressed until negative energy balance returns to balance. Similar to our ﬁndings,
Loefﬂer et al. (1999) reported cows with a BCS of 3.0 had a trend for increased PR. It
appeared that cows with extreme body condition scores either (very thin or fat) tended to
have a lower PR/Al. One explanation for this is that cows over or under conditioned are
more susceptible to metabolic problems, infections and reproductive difﬁculties
(Gearhart et al., 1990).

Previous calving difﬁculty scores (Dematawewa and Berger, 1997) for each cow

included in the study were recorded and evaluated for potential impact on subsequent

68

 

fertility. We did not discover a signiﬁcant difference in fertility in cows without calving
difﬁculty when compared with cows with slight to severe difﬁculty. However, numbers
of animals with extreme calving difﬁculty were too few to accurately determine the effect
of extreme calving difﬁculty on fertility.

Considerable work has been dedicated to studying the relationship of milk
production and fertility and contradictory evidence is available in the literature. Some
studies report a negative effect of high milk production on fertility (Faust et al., 1988;
Fonseca et al., 1983; Laben et al., 1982; Ron et al., 1984; Spalding et al., 1974) while
others found no effect or slightly positive results (Boyd etal., 1954; Eckles, 1929; Everett
et al., 1966; Gaines, 1927; Hillers et al., 1984; Peters and Pursley, 2002). Our results
would favor the latter. Data from current study is consistent with ﬁndings that cows with
above-average milk production had a greater PR compared with lower producing cows
(Peters and Pursley, 2002). In the current study, second lactation cows with above
average milk production had a 15 % difference in PR/AI over their below average
contemporaries. This is different from the results of Peters and Pursley (2002) who
reported that ﬁrst parity cows with above average milk production had the greatest
advantage in fertility over below average contemporaries. Badinga et al. (1985)
observed poor-producing cows to be less likely to conceive and more susceptible to
health-related problems. Thus, high producing cows may have fewer health problems,
such as lameness, mastitis, RP or ketosis.

SCS are routinely used as an indicator of subclinical mastitis. Mastitis can cause
severe pain, loss of milk production and stress on the cow. Mastitis has previously been

associated with poor reproductive performance (Dobson et al., 2001). However, in the

69

current study we saw no difference in PR in cows with low SCS compared to cows with
high SCS. Again, over interpretation of these results are discouraged as we did not have

a lot of variability in SCS for cows, with very few showing clinical signs of mastitis.

70

CONCLUSION

In conclusion, more female calves were generated with timed insemination at 32
to 40 h before ovulation with a 10 % decrease in fertility. Site of semen deposition did
not provide a viable technique to maintain fertility for inseminations that occurred at 32
to 40 h before ovulation. More effort needs to be dedicated to establishment of an
effective regime that incorporates AI prior to ovulation and maintains fertility. Selection
of high fertility sires may be a viable option. The sire in the current study with the
highest PR/AI was unaffected by the extended time of his semen in the uterus before
fertilization. Surprisingly, this sire had higher fertility in the -8 h groups, which
presumably challenged sperm survivability to a greatest extent than the 16 h group.
Timing of AI prior to ovulation may be a novel ﬁeld test to determine fertilization

capacity of service sires.

Some of the physiology and management factors evaluated in this study proved to
have an impact on PR/AI in lactating dairy cows. We discovered that multiple
ovulations, large follicle size and small CL size at the time insemination are signiﬁcantly
associated with increased fertility. Additionally, pregnancy 1055 appeared to be more
closely associated with physiological factors rather than management. It was discovered
that cows with CL > 17 mm at the time of insemination had more pregnancy loss than
cows with smaller CL. Cows with a single ovulation had more pregnancy loss than cows

with multiple ovulations. Future work to determine mechanisms involved in pregnancy

71

loss should be pursued. Pregnancy loss may be an important link to the decline in

fertility in multiparous lactating dairy cows.

72

 

BIOGRAPHY

Melissa Wolf Macfarlane is the fourth generation of her father’s family to be born and
raised in southern Oregon. She graduated from Crater High School in Central Point,
Oregon in 1996. Following graduation she left her roots and moved to California. In the
fall of 2000 she earned a Bachelor of Science degree from California State University,
Chico. In the summer of 2001 she accepted an assistantship with the Department of
Animal Science Michigan State University. In the fall of 2003 she received her Master of
Science Degree. Following degree completion she returned to California to pursue a

career in post-secondary agriculture education.

73

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