ha y... t a ”hi. :0- h w. -‘a. ,""1. ,W 73.13“" . ~ ‘ “flak. w v .. “a", {.3 "- ... w. ' ‘ “Lsfi‘L‘kuh-m‘CT-ix‘u‘: m" can 35 ---,\.M-_.w ”mtfggu‘ awry; ii Manna-‘rwy: » «4 x . . 1 "Mr. .m:'-‘".l) (Gill, 1978). Means were compared using Student's t test applied to a set of orthogonal contrasts (controls vs. rbGRF and rbST; rbGRF vs. rbST) for both treatment averages and within-period comparisons, (Gill, 1978). Bonferroni t test was used when more specific mean comparisons were needed. The criterion for statistical significance was P<. 1; therefore, any comparisons in which P value was greater than .1 was designated "not significant" (NS). RESULTS Somatotropin and pituitary measurements Somatotropin concentrations in serum averaged across d 1, 29 and 57 were 18.9, 19.2 and 3.1 ng/ml for rbGRF, rbST and controls, respectively (Figure 2). There was a treatment by time interaction (P<.01); therefore, comparisons between treatment means were performed within each sampling day (d 1, 29 or 57). Serum ST concentrations were increased for rbGRF and rbST in comparison with controls on d l, 29 and 57 (P<.01). On experimental d 1, ST concentration in serum for rbST-infused cows was higher than for rbGRF-infused cows (P<.01), but rbGRF-infused cows had elevated ST concentration in serum on d 29 (P<.05) as compared with rbST-infused cows. There was no difference in ST concentration in serum between rbGRF- and rbST- infused cows on experimental d 57 (P=.l3). Pituitaries from rbGRF-infused cows were heavier than those of controls (P<.01) and those of rbST-infused cows (P<.02; Table l). Somatotropin content of pituitaries from rbGRF-infused cows was numerically lower than rbST-infused cows, but it was significantly lower than controls (P<.01). Somatotropin concentrations in pituitaries from both control (P<.01) and rbST-treated cows (P<.01) were greater than that of rbGRF-treated cows. Milk yield and composition Solids-corrected-milk yield from d -14 to 0 (pre-treatment) was significant when tested as a covariate (P<.l); therefore, SCM yields were adjusted using 26 27 N U! l N G I Somatotropin, ng/ml ' l i l 10 r- 5 r- <% 9 4,) 0 l l l 29 57 Experimental day Figure 2. Least squares means of concentrations of somatotropin in serum of cows receiving no treatment ( -e— ), 12 mg rbGRF/d ( +) or 29 mg rbST/d ( + ). Standard error of the difference among treatments was 1.97 ng/ml on d 1 and 29, and 2.07 ng/ml on d 57, standard error of the difference of periods within a treatment was 1.6 ng/ml. Mean comparisons of periods within a treatment were performed using Bonferroni t test ( n=9 or 10). 28 Table 1. Least squares means of weight, ST content and ST concentration in anterior pituitary glands of 'cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST). Mean comparisons were performed using Bonferroni t test (n=10). P values CON CON GRF vs. CON GRF bST SED vs. bST vs. GRF bST Pituitary 2.0 2.6 2.1 .2 NS .01 .02 weight, g ST content, mg 35.0 21.3 30.2 4.1 NS .01 NS ST 17.9 8.3 14.7 1.9 NS .01 .01 concentration, Ins/g 29 pre-treatment milk yields as a covariate. Averaged throughout the experiment, SCM yields of rbGRF (33.3 kg/d) and rbST (34.1 kg/d) were increased (P<.01) relative to controls (29.1 kg/d) (Figure 3). There was no difference in SCM yields between rbGRF- and rbST- infused cows . Pre-treatment milk fat, lactose, and protein percentages were significant when tested as covariates (P<. 1); therefore, each milk component was adjusted using their respective pre-treatment values as a covariate. There was no difference in percentage of any milk component between any of the treatments (Table 2). Somatic cell count values were converted to natural logarithms (log SCC) in order to minimize heterogeneous variance. However, there was no difference in log SCC among treatment groups. Body weight, dry matter intake, calculated energy balance, organ weights and carcass composition measurements Body weight values were adjusted using pro-treatment body weight as a covariate. There were no differences in average BW throughout the experiment among rbGRF (539.3 kg), rbST (542.1 kg) and control cows (536.5 kg) (Figure 4). However, slopes for BW throughout the experiment were positive and significant for controls (P<.01; adjusted r2 = .97), and rbGRF-treated cows (P<.03; adjusted r2 = .78). The body weight of one rbST-treated cow decreased from 564.5 (d 44,45) to 508.5 kg (d 58,59) due to lack of feed intake associated with a case of acute mastitis. Although that animal did not test as an outlier (Gill, 1978), if she was removed from the analysis, BW average for the rbST group increased from 542.1 to 546.1 kg on d 58,59, and the slope for BW throughout the experiment approached significance (P=.06; adjusted r2 = .67). Means for DMI were adjusted using pretreatment DMI as a covariate. Averaged throughout the experiment, DMI was not different among treatments 30 36- Solids corrected milk (kg/d) b) b) m In) M U G I-I N be vb M l I F I l l \ N \o l 28 l l l I I I I I 1-7 8-14 15-21 22-28 29-35 36-42 43-49 50-56 57-63 Experimental day figure 3. Least squares means of solids corrected milk yield of cows receiving no treatment (—e— ), 12 mg rbGRF/d ( + ) or 29 mg rbST/d ( + ). Each point represents the average of a treatment group within each 7-d period, adjusted by covariance using pro-treatment SCM as a covariate. Standard error of the difference among treatments was 1.0 kg/d (n=10). 31 Table 2. Least squares means of percentages of milk components of cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST). Values represent the average of cows sampled once per week in each treatment group, adjusted by covariance using pre-treatment milk composition as a covariate (n=10). CON GRF bST SED Fat, % 3.2 3.3 3.4 .15 Lactose, % 5.0 5.0 5.0 .03 Protein, % 3.0 3.1 3.0 .04 log SCC 4.7 4.4 4.5 .3 32 550 - K UI l K O I \\ u Body weight (kg) Us I» u. I 530 r 525 1 1 r 1 2, 3 l6, 17 30, 31 44, 45 58, 59 Experimental day Figure 4. Least squares means of body weight of cows receiving no treatment ( —e— ), 12 mg rbGRF/d ( +) or 29 mg rbST/d ( + ). Standard error of the difference among treatments was 4.98 kg. Means were adjusted by covariance using pro-treatment body weight as a covariate (n=10). 33 (Figure 5). However, slopes for DMI throughout the experiment were positive and significant for controls (P<.01; adjusted r2 =.86), rbGRF-treated cows (P<.01; adjusted r2 =.80) and rbST-treated cows (P<.01; adjusted r2 =.73). Within each treatment group, means of each experimental period were compared with means from d 1-7. For rbGRF- treated cows DMI was increased in relation to d 1-7 on all other days (P<.1). For rbST- treated cows DMI tended to increase on d 43-49 and 57-63 (P<.l). For controls, DMI was elevated on d 43-49 and 57-63 (P<.05). On d 57-63 DMI tended to be greater for rbGRF than for rbST-treated cows (P<.1). Change in DMT from d 1-7 to (1 8-14 were not statistically different among treatments (P>.1). Means for calculated BB were adjusted using pre-treatment calculated EB as a covariate. There was a treatment by time interaction on calculated EB means (P<.01); therefore, comparisons between treatments were performed within each period. Calculated energy balance was lower for rbGRF- and rbST-treated cows in comparison with controls from d 17 to d 59 (P<.01); (Figure 6). On (1 59 calculated EB was greater (P<.05) for rbGRF- as compared with rbST-treated cows. For rbGRF-treated cows, calculated EB on d 31 was lower (P<.01) than on d 59. Cows treated with rbST tended to have lower calculated EB from d 17 to d 45 (P<.1) as compared with d 3. Controls had elevated calculated EB on d 45 and 59 (P<.01) as compared with d 3. Carcass weight was significant (P<. 1) when tested as a covariate for weights of heart ventricles, intestine, kidney, liver, lung and spleen; therefore, weights of these organs were adjusted by covariance using carcass weight. Weights of heart ventricles, intestine, kidney, lung and spleen were greater for rbGRF- and rbST-infused cows as compared with controls (Table 3). Because liver weight means of rbGRF and rbST groups were dissimilar, means were not compared as orthogonal contrasts. Instead non- orthogonal comparisons were performed by Bonferroni t test. Cows infused with rbST 34 24 P "3 23 9 3 g V E .5 22 is f. E t: a 21 20 I I I J I I I I I 1-7 8-14 15-21 22—28 29-35 36-42 43-49 50-56 57-63 Experimental day Figure 5. Least squares means of dry matter intake of cows receiving no treatment ( -e— ), 12 mg rbGRF/d ( +) or 29 mg rbST/d ( + ). Standard error of the difference among treatments was .42 kg/d, standard error of the difference of periods within a treatment was .51 kg/d, standard error of the difference of treatments within a period was .6 kg/d. Mean comparisons of periods within a treatment and treatments within periods were performed using Bonferroni t test ( n=10). 35 10r- 0 Energy balance, Mcal/d at u 4- / 1 r 1 1 m 2 3 17 31 45 59 Experimental day Figure 6. Least squares means of calculated energy balance of cows receiving no treatment (-9— ), 12 mg rbGRF/d ( + ) or 29 mg rbST/d (+ ). Standard error of the difference among treatments within a period was 1.5 Mcal/d, standard error of the difi'erence of periods within a treatment was .87 Meal/d. Mean comparisons of periods within a treatment were performed using Bonferroni t test ( n=10). 36 Table 3. Least squares means of organ weights of cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST). Means were adjusted by covariance using carcass weight as a covariate (n=10). P values CON vs. GRF vs. CON GRF bST SED GRF and bST bST Heart ventricles , 2.0 2.1 2.1 .08 .05 NS kg Intestine, kg 8.8 10.1 9.9 .48 .02 NS Kidney, kg 1.5 1.6 1.6 .06 .05 NS Liver, kg 9.3 9.7 10.6 .28 "‘ * Lung, kg 3.5 3.9 4.0 .14 .01 NS Spleen, kg 1.0 .9 1.0 .05 NS NS * Liver weight means were compared using Bonferroni t test. The comparisons performed were: controls vs. rbGRF (NS), controls vs. rbST (P<.01) and rbGRF vs. rbST (P<.02). 37 had greater liver weights than those of rbGRF-infused and control cows (P<.01), but rbGRF treatment did not increase liver weight in relation to untreated control cows. Body weights at slaughter were not different among the three treatment groups (Table 4). ‘ Carcass weights tended to be greater (P<.08) for control cows as compared to rbGRF- and rbST-infused cows. Similarly, dressing percentage was greater (P<.02) for controls than for rbGRF- and rbST-treated cows. Carcass water percentage, kg of carcass water and carcass water accretion were greater for rbGRF- and rbST- infused cows as compared with controls (P<.01). Similarly, carcass protein percentage was greater (P<.02), and kg of carcass protein and carcass protein accretion tended to be greater (P=.06) for rbGRF- and rbST- infused cows in relation to controls. In contrast, carcass fat percentage, kg of carcass fat and carcass fat accretion were greater (P<.01) for controls as compared with rbGRF- and rbST-treated cows. There were no differences in any of the carcass parameters between rbGRF- and rbST-treated cows (P>.1). Fat mobilization measurements Pre-treatment BCS was significant (P<.1) when tested as a covariate for BCS, but there was a significant treatment by covariate interaction (P<. 1); therefore, no adjustment was performed on treatment means. There were no overall treatment differences in BCS, although BCS tended to be lower (P<.l) for rbGRF- and rbST- treated cows in relation to controls on d 59 of the experiment (Figure 7). Both rbGRF (279.5 qu/L) and rbST (284.7 qu/L) treatments increased NEFA concentration in serum of cows as compared with controls (196.9 qu/L) (P<.01) (Figure 8). Carcass weight was significant (P<.1) when tested as a covariate; therefore, means of omental and perirenal fat weights were adjusted by covariance. Both rbGRF- and rbST-infused cows had lower omental fat weights and omental fat accretion as 38 Table 4. Least squares means of body weight at d 63 and carcass parameters of cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST). Means of kg and accretion of carcass water, protein and fat were adjusted by covariance using carcass weight as a covariate (n=10). P values CON vs. GRF vs. CON GRF bST SED GRF and bST bST Body weight, kg 547.2 539.9 536.9 14.3 NS NS Carcass, kg 241.5 233.5 226.5 7.1 .08 NS Dressing, % 44.14 43.31 42.18 .65 .02 NS Carcass water, % 67.9 71.5 71.8 .6 .01 NS Carcass water, 159.1 167.2 167 .2 1.4 .01 NS kg Carcass water -.01 .12 .12 .022 .01 NS accretion, kg/d a Carcass protein, 18.2 19.1 19.4 .4 .02 NS % Carcass protein, 42.8 44.6 45.1 1.1 .06 NS kg Carcass protein .01 .04 .05 .02 .06 NS accretion, kg/d a Carcass fat, % 1 1.9 7.8 6.6 .9 .01 NS Carcass fat, kg 27.5 18.4 16.4 2.1 .01 NS Carcass fat -.008 -.15 -.18 .03 .01 NS accretion, kg/d a Value at d 181 minus value at d 118 divided by 63 d. 39 2.5 " E 3 ‘ r. i: ‘3. ~ g c 35 a 24 ' 8 >5 0 u '5 § \’ 2.3 L I l I 3 17 31 45 59 Experimental day Figure 7. Least squares means of body condition score of cows receiving no treatment (-9- ), 12 mg rbGRF/d ( +) or 29 mg rbST/d ( + ). Standard error ofthe difference among treatments was .1. Standard error of the difference among treatments within d 45 and within d 59 was .1 (n=10). 40 320 l- l E 280 , ii a 260 b E h- B 240 - E E» if: 220 - a O z 200 - G— 180 I T? I 1 29 57 Experimental day Figure 8. Least squares means of concentration of serum NEFA of cows receiving no treatment ( —e— ), 12 mg rbGRF/d ( + ) or 29 mg rbST/d ( + ). Standard error of the difference among treatments was 27.7 qu/L (n=10). 41 compared with controls (P<.01; Table 5). Similarly, perirenal fat weight and perirenal fat accretion were lower for rbGRF- and rbST-infused cows relative to controls. In contrast, there was no difference among treatment groups in the 12th rib fat depth of COWS. Mammary function measurements Mammary parenchymal weight was not different among the three treatment groups (Table 6). However, when expressed per 100 kg of carcass weight, parenchymal weight tended (P=.07) to be greater for rbGRF- and rbST-treated cows as compared with controls. The rbGRF- and rbST-infused cows had 34% less intraparenchymal fat than controls (P<.01). There was no difference in mammary parenchymal weight between rbGRF- and rbST-infused cows. Total DNA, DNA concentration, and DNA accretion were not different among treatment groups (Table 7). In contrast, total RNA (P<.05), RNA concentration (P<.01), RNA accretion (P<.01), and RNA to DNA ratio (P<.05) were greater for rbGRF- and rbST- infused cows as compared with controls. There was no difference in any of the measures of RNA between rbGRF- and rbST-infused cows. There was no difference in the ratios of SCM per kg of parenchyma or per g of DNA among the three treatment groups in this experiment (Table 8). Lactose synthesis rate of mammary parenchymal tissue slices was not different among treatment groups (Figure 9). 42 Table 5. Least squares means of adipose tissue weight of cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST). Means of kg and accretion of omental and perirenal fat were adjusted by covariance using carcass weight as a covariate (n=10). P values CON vs. GRF vs. CON GRF bST SED GRF and bST bST Omental fat, kg 5.2 2.9 2.4 .42 .01 NS Omental fat 2.21 -33.8 -42.4 6.05 .01 NS accretion, g/d a Perirenal fat, kg 3.5 1.6 1.4 .42 .01 NS Perirenal fat a .75 -30.4 -33.05 6.5 .01 NS accretion, g/d 12th rib fat depth, 1.2 1.1 1.1 .2 NS NS cm a Value at d 181 minus value at d 118 divided by 63 d. 43 Table 6. Least squares means of mammary parenchymal weight and intraparenchymal fat of cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST) (n=10). CON GRF P values bST SED CON vs. GRF vs. GRF and bST bST Mammary parenchymal .77 .83 weight a Mammary parenchymal .32 .36 weight b Intraparenchymal fat 6 31.8 20.1 .85 .07 NS NS .37 .03 .07 NS 21.8 2.5 .01 NS a kg dry, fat-free, solids-non-fat-corrected parenchyma per 1/2 gland b kg dry, fat-free, solids-non-fat-corrected parenchyma per 1/2 gland per 100 kg carcass Wt. c g/ g dry, fat-free, solids-non-fat—corrected mammary parenchyma. Table 7. Least squares means of mammary parenchymal nucleic acid composition of cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST) (n=10). P values CON GRF bST SED CON vs. GRF vs. GRF and bST bST Total DNAal 22.7 24 23.6 2 NS Ns b Total DNA 9.4 10.3 10.5 .8 Ns Ns DNA concentration c 29.2 29.0 28.1 1.0 Ns Ns DNA accretion d -.008 .006 .008 .01 NS Ns Total RNA 3 60.8 77.7 73.7 6.4 .02 Ns b Total RNA 25.3 33.4 32.5 2.5 .05 NS C RNA concentration 79.4 93.6 88.1 4.6 .01 NS d RNA accretion -.02 .1 .09 .04 .01 NS RNA/DNA 2.7 3.2 3.2 .2 .05 NS 3 g per 1/2 gland b g/ 100 kg carcass wt per 1/2 gland. c mg/g dry, fat-free, solids-non-fat-corrected mammary parenchyma. d Value at d 181 minus value at d 118 divided by 63 d (g/d). 45 Table 8. Least squares means of solids corrected milk yields from d 56 to 62 expressed per kg of mammary parenchyma and per kg of mammary parenchymal DNA for cows receiving no treatment (CON), 12 mg rbGRF/d (GRF) or 29 mg rbST/d (bST) (n=10). P values CON GRF bST SED CON vs. GRF vs. GRF and bST bST SCM/kg parenchyma a 22.7 24 23.6 2 NS NS SCM/gDNA b 9.4 10.3 10.5 .8 Ns NS a kg/kg dry, fat-free, solids-non-fat-corrected parenchyma per 1/2 gland. b kg/g total DNA per 1/2 gland. 46 1.2 " Lactose synthesis, mg/h/g of tissue as I Figure 9. Least squares means of lactose synthesis of cows receiving no treatment (open bar), 12 mg rbGRF/d (solid bar) or 29 mg rbST/d (dashed bar). Standard error of the mean for each treatment is depicted on top of each bar (n=6 to 9). DISCUSSION Galactopoietic effects of rbGRF and rbST have been profusely documented for dairy cattle (Dahl et al., 1991; Dahl et al., 1993; Bauman, 1992; Bauman and Vernon, 1993). Therefore, as expected, in the current study both rbGRF and rbST treatments increased SCM yield of cows compared with non-infused controls. Elevated milk yield was probably largely due to elevated ST concentration in serum, caused by rbGRF- stimulation of endogenous ST secretion from the anterior pituitary gland, or by exogenous rbST itself. Dahl et al. (1993) infused lactating dairy cows for 60 d with the same doses of rbGRF and rbST used in the current experiment and reported a 10 % greater increase in SCM yield for rbGRF- as compared with rbST-treated cows during the last 20 d of infusion. This disagrees with data from the current experiment, where SCM yields were similar between rbGRF- and rbST-infused cows throughout the experiment. Several reasons could possibly explain this discrepancy. For example, animals in the present experiment were milked three times daily which is by itself galactopoietic (DePeters et al., 1985). Moreover, Bauman (1992) stated that milk yield response to increasing doses of rbST increases linearly until a plateau is reached at a dose of 30 to 40 mg of rbST/d. Thereafier, milk yield is only increased marginally, even with several fold higher doses of rbST. Speicher et al. (1993) reported the additive effects of thrice (vs. twice) daily milkings and rbST injections on milk production of cows. Therefore, it is possible that effects of three daily milkings added to the effects of elevated ST in serum maximized the galactopoietic response of cows in the present experiment to ST (i.e., a greater 47 48 concentration of ST in serum would not further stimulate milk production). In contrast, cows in the experiment of Dahl et al. (1993) were only milked twice daily. In addition, at the beginning of the experiment their cows were in a later stage of lactation (175 d). Furthermore, the majority of their cows were multiparous. Collectively, the cows used in the experiment of Dahl et al. (1993) may not have been as close to maximum production of milk as those used in the present study. I speculate, therefore, that their cattle may have been more suitable to show differences in milk yield response to rbGRF vs. rbST as compared with the cattle in my experiment. Treatment of dairy cows with rbST does not affect percentage of milk components (Bauman, 1992). Similarly, Dahl et al. (1990) reported that rbGRF infusions did not change gross composition of milk. We also found no difference in percentages of any milk component among treatments. Therefore, since total yield of milk increased, total yields of lactose, fat and protein were elevated for rbGRF- and rbST-treated cows relative to controls. An immediate result of rbST administration is increased radioimmunoassayable levels of ST in serum (Dahl et al., 1991 and Dahl et al., 1993). Similarly, administration of rbGRF acutely and specifically increases concentration of ST in serum (Enright et al., 1986). Moreover, prolonged continuous infusion of rbGRF maintains elevated concentrations of ST in serum for at least as long as 90 d (Tucker et al., 1993; personal communication). Dahl et a1. (1993) noticed a greater increase in milk production for cows infused with rbGRF as compared with cows infused with rbST, although the concentrations of ST in serum for both groups were similar. Possibly, the galactopoietic effects of rbGRF are not solely mediated by ST. In the present experiment, my aim was to increase concentrations of ST in serum to similar levels for both rbGRF and rbST treatment groups. I wanted to examine potential ST-independent effects of rbGRF on variables involved in galactopoiesis. Both rbGRF and rbST 49 treatments increased concentrations of ST in serum of cows in relation to untreated controls on d l, 29 and 57 of the experiment. However, ST concentrations in serum were greater for cows treated with rbST than for cows treated with rbGRF on d 1. In contrast, ST concentrations were greater for cows treated with rbGRF on d 29 and there was a tendency for ST concentrations to be greater on d 57 as compared with cows infused with rbST. This does not agree with the results of Dahl et al. (1993). In their experiment they infused the same doses of rbGRF and rbST as used in the present study and achieved similar concentrations of ST in serum between both groups. Concentrations of endogenous ST in serum of cows decrease with age (Lapierre et al., 1992), probably reflecting a progressive diminution of pituitary synthetic capacity (Sadow and Rubin, 1992). Indeed, Dahl et a1. (1993) worked mostly with multiparous cows (75 % of the animals in that experiment were multiparous) and average concentrations of ST in serum of both rbGRF-treated cows (12.5 ng/ml) and also control cows (1.2 ng/ml) were numerically smaller than concentrations in cows of the current experiment (18.9 and 3.1 ng/ml, for rbGRF-treated and controls, respectively). It should be noted that the assay used for ST was the same for both experiments. Therefore, a possible explanation for the difference in serum concentrations of ST between rbGRF- and rbST-infused cows of the present study may be that primiparous cows in the present experiment were more responsive to stimulation by rbGRF than the predominantly multiparous cows used in the experiment conducted by Dahl et al. (1993). As a result, ST concentrations in serum were elevated for rbGRF- relative to rbST-infused cows on d 29 and 57. Therefore, if a smaller dose of rbGRF had been used in the present experiment, probably similar concentrations of ST in serum would have been achieved throughout the experiment for both rbGRF- and rbST-infused cows. But whether such differences in ST concentrations in serum would be biologically significant is questionable. Eppard et al. (1985) showed that the milk yield response increased in a curvilinear fashion with linear increases in ST 50 concentration in serum, tending to plateau between 15 and 20 ng/ml of ST. Moreover, as indicated previously, cows in the experiment conducted by Dahl et al. had lower concentrations of ST in serum than cows in the current experiment, even though the doses used in both ‘ experiments were the same. However, milk production response to hormone treatments in their experiment were within the expected range (approximately 35% above the controls), suggesting that those lower concentrations of ST in serum sufficed to evoke significant galactopoiesis. I speculate that ST concentration in serum of rbGRF- and rbST-treated cows in the present experiment were at the plateau region of the curve of milk response to ST concentration in serum; therefore, the fact that ST concentrations in serum were not similar throughout the experiment should not cause different milk production responses between these two groups of cows. In fact, milk production was similar between rbGRF- and rbST-infused cows, therefore, differences in ST concentration in serum were probably not biologically significant, at least in terms of milk production. Concentration of ST in serum is not the sole indication of stimulation of the cascade of events promoted by infusions of rbGRF and rbST. In the same experiment described in my thesis, VanderKooi (1993) reported that both rbGRF and rbST treatments increased concentrations of IGF-I and IGFBP-3 in serum and IGF-1 mRNA abundance in liver in relation to controls. However, IGF-I and IGFBP-3 concentrations in serum and IGF-1 mRNA abundance in liver were greater for rbST- relative to rbGRF- treated cows. Despite the above described dissimilarities in variables influencing the somatotropic cascade, milk yields were not different between rbGRF- and rbST-infused cows. Therefore, VanderKooi (1993) suggested that rbGRF exerted at least part of its galactopoietic effects through processes not mediated by IGF-I nor IGFBP-3. Another interpretation for the similar milk production for rbST- compared with rbGRF-treated cows, despite the greater levels of IGF-I and IGFBP-3 for rbST-treated cows, is that, 51 possibly, levels of IGF-I and IGFBP-3 were above the threshold for milk synthesis stimulation in rbGRF- and rbST-treated cows. Therefore, greater stimulation of such variables by rbST did not result in increased milk yield compared with rbGRF. Frohman et al., (1992) reported in rats that treatment with exogenous GRF increased anterior pituitary weight, and this was associated with hyperplasia of somatotrophs. Such an increase in anterior pituitary weight was also observed in rats transgenic for GRF (Asa et al., 1990). The increased anterior pituitary weight of rbGRF- treated cows in the current experiment, relative to controls, could indicate proliferation of somatotrophs, but I do not have a direct measure of this possibility. Effects of GRF on synthesis and release of ST from somatotrophs have been widely documented (Padmanabhan et al., 1987 and Frohman et al., 1992). Elevated concentrations of ST in serum of cows infused with rbGRF are probably a result of increased synthesis and release of ST from somatotrophs. Indeed, content of ST in anterior pituitaries of rbGRF- treated cows was smaller than that of controls, which I speculate supports the notion that release of ST from somatotrophs may have been increased. However, rbGRF-stimulated synthesis of ST from somatotrophs was probably also elevated because serum concentration of ST remained elevated throughout the experiment. Thus, I have no evidence that rbGRF caused the pituitary to become refractory to rbGRF treatment over the 63-d period of this experiment. The similarities between rbST-infused and control cows for pituitary weight and ST content and concentrations suggests that rbST did not cause refractoriness to inhibit synthesis or release of ST from the anterior pituitary gland. Knight et al. (1990) reported increased mammary gland volume in goats treated with ST. They attributed such results to decreased mammary cell loss and increased mammary cell volume. In contrast, Capuco et al. (1989) reported no changes in mammary DNA in lactating dairy cows in response to rbST. In the current 52 experiment, rbGRF and rbST treatments each tended to increase mammary parenchymal weight relative to controls. However, this result could not be explained by a change in mammary cell number because treatments did not affect total DNA nor DNA concentration in mammary tissue. I suggest that increased cell size may have accounted for the increased parenchymal weight. Total RNA is an index of cell metabolic activity. Baldwin (1990) reported increased total RNA per mammary gland in lactating cows treated with rbST. In the current experiment there was an increase in mammary tissue total RNA, RNA concentration, RNA accretion between d 118 and 181 of lactation and RNA to DNA ratio for cows treated with rbGRF and rbST. Data from Baldwin (1990) suggest that rbST increased the secretory capacity of the mammary gland. These findings support the argument that at least part of the action of rbGRF and rbST on galactopoiesis is through an effect on metabolism within the mammary gland. However, I was unable to confirm such an increase in metabolic efficiency in the mammary tissue when SCM was expressed on the basis of kg of parenchyma or per g of DNA. Perhaps, calculation of these ratios diluted effects of treatments previously observed. In contrast to the evidence of increased secretory activity in mammary cells was the finding that rbGRF or rbST treatments did not affect lactose synthesis in incubated mammary tissue slices. Increased RNA levels per mammary cell reflects increased levels of transcription and eventually translation of proteins involved in milk synthesis. Therefore, cells possessing more synthetic machinery should have had greater ability to transform glucose into lactose. The reason for this lack of effect of rbGRF and rbST on lactose synthesis is unknown. An important characteristic of the lactating cows used in the present experiment is the fact that these animals were still growing. This situation is of interest because in the overall scheme of nutrient partitioning, body growth was not impaired as a 53 consequence of the great demand of nutrients by the mammary gland as a result of hormone treatments. These growing cows treated with rbGRF and rbST may have adopted different strategies to support milk synthesis than mature cows that are not growing. Some examples are discussed below. Dry matter intake gradually increases as a result of rbST treatment (Bauman, 1992). This is in contrast to the data from Dahl et al. (1993), in which DMI decreased within time in rbGRF-infused, rbST-infused and control cows. In the current experiment, DMI increased for all groups throughout the 63-d of the experiment. Two possible explanations for this finding are that in the experiment of Dahl et a1. (1993) the majority of the cows were multiparous (i.e., skeletal growth had probably ceased), and they started the experiment in a more advanced stage of lactation (175 d). Therefore, their cows may not have had as great a demand to consume feed as did the growing and high producing cows of the present study. I also found that DMI began to increase by the second week after treatment started for cows treated with rbGRF, and gradually increased for both rbST-treated and control cows. However, this apparently quicker increase in DMI for rbGRF-treated cows was not statistically significant. 1 speculate that rbGRF-treated cows may have relied more on increased DMI to support increased milk production than rbST-treated cows. Effects of rbGRF and rbST on carcass composition also support the concept that these hormones were simultaneously affecting grth and lactation of cows in the current experiment. Several studies indicate that rbST treatment changes carcass composition of growing animals: increasing lean (protein and water) and decreasing fat in the carcass (Enright, 1989; Moseley et al., 1992; Vestegaard et al., 1993). In fact, Moseley et al. (1992) showed that an important effect of rbST injected to feedlot steers was that it promoted changes in the composition of gain (i.e., increased lean, decreased fat tissue) rather than improving average daily gain. In contrast, for mature lactating 54 Holstein cows, rbST treatment did not affect lean tissue growth (Solderholm et al., 1988). Treatments with rbGRF and rbST increased carcass water and protein and decreased carcass fat of cows in the current experiment. Accretion of lean tissue is consistent with the fact that animals in the present experiment were still growing, as evidenced by increasing BW in all groups. Therefore, primiparous cows in the present experiment responded to the effects of rbGRF and rbST more like growing steers than like mature cows. Mobilization of adipose depots probably provided the extra energy needed for milk synthesis and lean tissue growth. Another possible strategy adopted by rbGRF- and rbST-treated cows in order to cope with simultaneous processes of grth and lactation could involve alterations in organ weights. Increased organ weights possibly play a role in increasing availability of nutrients to the mammary gland and thereby contribute to the galactopoietic effects of rbGRF and rbST. For example, Davis et al. (1988) reported an increase in cardiac output and mammary gland blood flow in cows treated with rbST. They interpreted this finding as a mechanism influenced by rbST that directed more nutrients to the mammary gland for milk synthesis. Increased cardiac output may explain increased weight of hearts (increased mass to support increased activity), lungs (more blood had to be oxygenated per unit of time) and kidneys (more blood had to be filtered per unit of time) in rbGRF- and rbST-treated cows in the current experiment. This contrasts with findings of Brown et al. (1989), who reported that rbST treatment did not affect weight of the above mentioned organs, but the cows used in their study were mature. In contrast, they reported that rbST treatment increased foregut mass, which was also observed in the current experiment. Increased weights of intestines of cows in the current experiment may reflect an increased ability of rbGRF- and rbST-treated animals to ingest and absorb nutrients. It is possible that increased organ weights were associated with the ability of 55 rbGRF- and rbST-infused cows in the present study to repartition nutrients towards the processes of growth and lactation. In addition to increased organ weights in response to rbGRF and rbST treatments, liver weights were increased in rbST-infused cows relative to controls. VanderKooi (1993) reported increased mRNA abundance for IGF-I in liver tissue of cows treated with rbST in relation to cows treated with rbGRF in the same experiment described in this thesis. Perhaps the recombinant bST used in this experiment stimulated liver tissue more than did endogenous ST secreted by the anterior pituitary gland of cows treated with rbGRF, resulting in greater liver weights for rbST- than for rbGRF-treated cows. Despite greater milk production for rbGRF- and rbST-treated cows compared with controls, DM1 and BW were similar among the three groups of cows throughout the entire experiment. The fact that control cows were ingesting as much feed as hormone- treated cows suggests that control animals were using feed nutrients in processes other than lactation. In fact, carcass weight tended to be greater and dressing percentage was also greater for controls relative to rbGRF- and rbST-infused animals, indicating that carcass grth was occurring in control cows whereas non-carcass grth was occurring in hormone-treated animals. Such carcass growth was mostly explained by increased fat deposition, as control cows had 12 kg more fat in the carcass than the average for hormone-treated cows (kg of carcass fat, unadjusted; data not shown). Unadjusted weights of organs were not different among the three groups of cows (data not shown), therefore, weights of organs measured did not account for non-carcass growth of hormone-treated animals. I speculate that weight of ingesta was greater in the gastrointestinal tract of both rbGRF- and rbST-treated cows relative to controls. Therefore, the similar body weights observed among the three treatment groups in this experiment had different origins; control cows increased their body weight mainly 56 because of increased deposition of fat in the carcass, whereas hormone-treated cows probably increased their body weight because of increased weight of ingesta in the gastrointestinal tract. Somatotropin is a homeorhetic regulator that influences metabolic processes by promoting a repartitioning of nutrients that favors preferential uptake of nutrients by the mammary gland during lactation (Peel and Bauman, 1987). Homeorhetic changes include increased mobilization and decreased accumulation of adipose tissue. However, these effects on adipose tissue are dependent on EB. For example, when lactating cows are in negative EB, lipid mobilization (lipolysis) is increased as reflected by decreased body fat and chronic elevation on serum concentration of NEFA (Barbano et al., 1992). In contrast, when cows are in positive energy balance, effects of ST on adipose tissue are on inhibition of lipogenesis, not on stimulation of lipolysis (Bauman, 1992). Data from the current experiment disagrees with such findings of Bauman. Calculated EB was always positive for all groups in all periods of the current experiment, but lipolysis was clearly stimulated in rbGRF- and rbST— infused cows. For example, there was increased NEFA concentration in serurrr, and decreased weights of omental, perirenal and carcass fat depots and decreased percent fat in mammary gland. Moreover, while control cows gained adipose tissue in both omental and perirenal depots, rbGRF- and rbST-treated cows mobilized fat from those same depots at a much greater rate than the gain of adipose tissue in control cows. These findings suggest that mobilization of fat was occurring. There are two possible explanations for the occurrence of lipolysis in cows in positive calculated EB. First, calculated EB does not necessarily reflect fat balance status. In fact, McNamara et al. (1986) discussed that although in positive EB, cows may be in negative fat balance, where mobilization of fat is greater than accretion of fat. Second, I speculate that it is not absolutely necessary that cows reach negative calculated 57 BB in order to initiate lipolysis. Perhaps, merely decreasing calculated BB is sufficient to evoke a lipolytic state. Liesman and Emery (1993, personal communication) collected adipose tissue from the omental depot of cows used in the current experiment and observed that lipoprotein lipase activity (an enzyme involved with accumulation of fat in adipose tissue) in rbGRF- and rbST-treated cows was reduced on d 63 (d of slaughter) as compared with controls. Moreover, they found less protein (i.e., more fat) per g of adipose tissue (omental depot) in rbGRF-infused cows in comparison with rbST-infused cows. Therefore, I postulate that although lipogenesis was equally impaired for both rbGRF- and rbST-treated cows, rbGRF-treated cows were mobilizing less fat from the omental depot compared with rbST-treated cows. In fact, weight of the omental fat depot was numerically greater for rbGRF- than for rbST-treated animals and concentration of NEFA in serum on d 57 was numerically smaller for rbGRF- relative to rbST-treated cows. Moreover, greater DMI and calculated EB on d 59 for rbGRF- compared with rbST-treated animals, leads me to speculate that towards the end of the experiment, rbGRF-treated cows were relying less on mobilization of adipose to maintain elevated milk production compared with rbST-treated animals. One main thrust of this thesis was to determine possible ST-independent effects of rbGRF on variables involved in galactopoietic responses. However, except for liver weight, there was no evidence for such effects in the variables that I measured. SUMMARY AND CONCLUSIONS The overall goal of this thesis was to examine potential mechanisms whereby rbGRF and rbST exert their galactopoietic effects in dairy cows. Infusions of rbGRF and rbST each increased concentrations of ST in serum of cows relative to non-infused controls. Milk yields were stimulated by rbGRF and rbST compared with no treatment. The first objective of this thesis was to compare the effects of rbGRF and rbST on homeorhetic adaptation. All three groups of cows increased BW, but composition of gain was different. For example, rbGRF- and rbST-treated animals had a greater percentage of lean tissue and smaller percentage of adipose tissue in the carcass relative to control animals. Increased organ weights, presumably to support increased cardiac output and repartitioning of nutrients, was also observed for both rbGRF and rbST groups relative to controls. Despite positive calculated EB for both rbGRF- and rbST-treated cows throughout the whole experiment, adipose tissue mobilization occurred as indicated by increased NEF A concentrations in serum and reduced weight of carcass, omental and perirenal adipose weights and mammary gland fat percentage. Mobilized fat probably provided extra energy necessary for increased milk production of cows treated with rbGRF or rbST. Altogether, homeorhetic changes set forth by rbGRF and rbST probably increased availability of nutrients to the mammary gland and skeletal muscle. My data support the notion that cows receiving rbST have a greater reliance on lipolysis as a source of energy for extra milk production than cows receiving rbGRF. A second objective was to examine the effects of rbGRF and rbST on mammary function. Relative to controls, rbGRF and rbST treatments did not affect 58 59 mammary cell numbers, but both treatments elevated metabolic activity of mammary cells. This indicates that both rbGRF and rbST treatments increase the ability of the mammary gland to take up milk precursors from the circulation and to synthesize milk. In contrast, lactosehsynthesis in vitro was unmodified by any of the hormone treatments in relation to controls. My third objective was to examine the effects of rbGRF and rbST on anterior pituitary function (i.e., synthesis and release of ST). Anterior pituitary weight, ST concentration in anterior pituitary and ST concentration in serum of rbGRF-treated cows support greater synthesis and release of ST than in controls. Both anterior pituitary weight and ST concentration in the anterior pituitary were similar when rbST-treated and control cows were compared. Development of refractoriness to either rbGRF or rbST did not occur. In addition, neither mammary function or body growth become refractory to either rbGRF or rbST. In conclusion, rbGRF and rbST increased the ability of dairy cows to repartition nutrients towards the mammary gland, and also increased the ability of the mammary gland to take up nutrients from the circulation and synthesize nrilk. None of the variables analyzed in this thesis provided strong evidence for galactopoietic effects of rbGRF independent of ST. APPENDICES APPENDIX A Extraction of ST from anterior pituitary glands Extraction of ST was performed on one half of each anterior pituitary gland of each animal. Frozen tissue was weighed, placed in a 16 x 100 mm plastic tube, minced with scissors, mixed with 1.0 ml of .01 N NaOH solution, and homogenized by sonication (Polytron, Brinkrnann, Westbury, NY). The pellet was rinsed with 2.0 ml of .01 N NaOH, centrifuged for 15 min at 1800 x g, and the supernatant was poured off and refrigerated. The steps were repeated with the remaining pellet, and the two supematants were combined for subsequent quantification of ST. 60 APPENDIX B Quantification of mammary parenchymal nucleic acids One part of frozen ground parenchyma (approximately 10 g) was placed with two parts of dry ice in the cup of a blender (Junke & Kunkel, West Germany) and blended until powdered, at 4 C. Powdered parenchyma was sifted through a flour Sifter, and the resulting material was stored at - 20 C for 6 to 8 h to allow the dry ice to sublimate. Approximately 1.0 g of powdered parenchyma was transferred to a 50 ml conical polypropylene tube (Corning Inc., Corning, NY). Forty ml of 100% ethanol was immediately added to the tube that was sealed with a screw cap. Tubes were placed horizontally in a rack and shaken vigorously for 24 h. Tubes were subsequently centrifuged for 20 min at 1000 x g and the supernatant was poured off. The same sequence was repeated using 40 ml of 2:1 methanolzchloroform followed by 40 ml of ethyl ether. After the ethyl ether was poured off, tubes were recapped and stored for 36 h at 4 C, so the residual ether would evaporate. Dry matter content of the resulting fat extracted powder was measured for use in calculations. A .14 g sample of frozen, fat extracted powder was used for DNA and RNA determinations, following the procedure of Tucker (1964). 61 APPENDIX C Adjustment of mammary parenchymal weight Dry, fat-free tissue weights of cows were normalized by estimating the amount of milk solids non-fat present per g of tissue (TSP/g) for cows killed at different times and subtracting that amount from dry, fat-free tissue weights. Calculations were performed for each cow as follows: tggl milk smthgsizgd pg h (TM/h ); TM/h = ( milk yield from AM milking for 3 d prior to slaughter) / 3 / 8.75 h (time elapsed between AM and midday milkings); t 11 on-ft th iz h /h' TS/h = (TM/h) x solids non-fat percentage from milk composition analysis (obtained 3 d before slaughter); to 11 nn-ft r ntinhlf rtti o l T TSS = (TS/h) x time elapsed between AM milking and slaughter time / 2; t._01non-ft- Ptfmd {J n-‘ ma: ' -.f_ e.' If --'/.‘ ' TSP/g = (TSS) / g of dry, fat free mammary parenchyma weight of half udder. 62 APPENDIX D Quantification of lactose synthesis In order to deproteinize samples, 500111 of media were mixed with 500,11 of 1 N perchloric acid and centrifuged for 10 min at 1550 x g, and immediately neutralized with 138111 of 4 N KOH solution. Tubes were than centrifuged for 10 min at 1550 x g to precipitate the salt, and placed in an ice bath for 15 min. Supernatants were saved for lactose quantification. Three hundred microliters of deproteinized samples were mixed with 1500 pl of potassium phosphate buffer (pH 7.5) and 200111 of .1 % NAD (Sigma Chemical Co., St. Louis, MO) solution. Absorbance (A1) at 340 nm wavelength was measured in a Beckman DU-64 spectrophotometer (Beckman Instruments Inc., Fullerton, CA) blank-calibrated with air. 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