Effects of recombinant bovine somatotropin on pregnancy per artificial insemination, corpus luteum cellular composition and endometrial gland morphometry in beef cattle

Theriogenology 141 (2020) 180e185

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Effects of recombinant bovine somatotropin on pregnancy per artificial insemination, corpus luteum cellular composition and endometrial gland morphometry in beef cattle
lia Paulozzi Costa a, Angela Gonella-Diaza b, Guilherme Pugliesi c, Nata ^ngela Bueno Cordeiro Maldonado d, Saara Carollina Scollari c, Maria ^ Castilho g, Barbara Piffero Mello c, Isabella Feltrin e, Renato Girotto f, Calie d , e, * Claudia Maria Bertan Membrive ~o Paulo State University - UNESP, Araçatuba, SP, 16015-050, Brazil Sa University of Florida, North Florida Research & Education Center, Institute of Food and Agricultural Sciences, Marianna, FL, USA c ~o Paulo, Pirassununga, SP, 13635-900, Brazil University of Sa d ~ Sao Paulo State University - UNESP, Dracena, SP, 17900-000, Brazil e ~ Sao Paulo State University - UNESP, Botucatu, SP, 18618-689, Brazil f
RG Advanced Genetics, Agua Boa, MT, 78635-000, Brazil g ~o Paulo West, Presidente Prudente, SP, 19050-920, Brazil University of Sa a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 February 2019 Received in revised form 11 September 2019 Accepted 16 September 2019 Available online 17 September 2019

The aim of this research was to evaluate the effect of recombinant bovine somatotropin (bST) on pregnancy per artificial insemination (P/AI), cellular composition of the corpus luteum (CL) and endometrial gland morphometry. In Experiment 1, Nelore cows (n ¼ 587) received a fixed-time artificial insemination (FTAI) protocol and, at insemination, received 0, 250 or 500 mg of bST subcutaneously (SC). In Experiment 2, Nelore cows (n ¼ 243) received 0 or 500 mg of bST, SC, on D7 (D0 ¼ day of FTAI). Blood samples were collected on D7 and D16 to measure progesterone (P4) concentrations. In Experiments 1 and 2, pregnancy diagnosis was performed 30 days after FTAI. In Experiment 3, Nelore heifers (n ¼ 20) received a FTAI protocol, but were not inseminated, and on D0 (ovulation day), they received 0 (bST 0; n ¼ 9) or 500 mg of bST (bST 500; n ¼ 11), SC. The heifers were slaughtered on D15 (D0 ¼ ovulation day), at which time the CL was evaluated for diameter, weight, a percentage of large (LLC) and small (SLC) luteal cells, and the concentration of progesterone in plasma measured. The number, perimeter and area of superficial and deep endometrial glands were evaluated. There was no difference in P/AI when bST was applied on D0 and D7. In Experiment 1, P/AI did not differ among treatments, with 59.28% (115/194), 58.38% (115/197) and 65.82% (129/196) for the bST 0, 250 and 500 treatments, respectively. In Experiment 2, P/AI did not differ between treatments, with 57.3% (71/124) and 60.5% (62/119) for the bST 0 and 500 treatments, respectively. Plasma progesterone concentrations on D16 was greater in the bST 500 (11.63 ± 0.84 ng/mL) than bST 0 (9.83 ± 0.88 ng/mL). In Experiment 3, there was no difference in ovarian diameter and weight, CL diameter, percentage of SLC, P4 concentrations and endometrial gland morphology. Heifers in the bST 500 treatment had heavier CL (3.11 ± 0.32 vs. 2.25 ± 0.20 g); however, the bST 0 treatment heifers had a greater percentage of LLC than did the bST 500 treatment (13.72 ± 1.16% vs. 8.60 ± 1.52). It was concluded that the doses of bST used in this study do not increase P/AI; however, they do cause changes in P4 concentration and the cellular composition of the CL. © 2019 Elsevier Inc. All rights reserved.

Keywords: bST Fertility Corpus luteum Endometrium Bos indicus

1. Introduction ~o Ribeiro de Barros (SP 294), * Corresponding author. Rodovia Comandante Joa ~o Paulo State University (UNESP), Dracena, SP, 17900-000, Brazil. Km 651, Sa E-mail address: [email protected] (C.M. Bertan Membrive). https://doi.org/10.1016/j.theriogenology.2019.09.023 0093-691X/© 2019 Elsevier Inc. All rights reserved.

The use of hormonal protocols for fixed-time artificial insemination (FTAI) provides a service rate of 100% without the need for estrous detection. In addition, FTAI induces cyclicity in females

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during the postpartum anestrus [1,2]. Despite the fact that FTAI is currently used on a large scale, the pregnancy per artificial insemination with this biotechnology in commercial beef herds in Brazil is 49.1% [2,3]. In dairy and beef bovine herds, early embryonic mortality is a major cause of reproductive failure [4,5]. In beef cows, fertilization rates normally range from 94.0 to 100% while the major portion of early embryo loss occurs after day 8 and before days 16e18 after FTAI. During this period, the embryos must elongate and produce interferon Tau to prevent luteolysis [6]. During this period, 20e40% embryonic losses are reported in the female zebu, most of which are attributed to the inability of the conceptus (embryo and the extra-embryonic membranes) to signal pregnancy recognition [7]. In bovine, the uterus and the embryo have receptors for growth hormone (GH, also known as somatotropin) and insulin-like growth factor (IGF-I), which, through endocrine, paracrine and autocrine actions, have important impacts during pregnancy establishment and maintenance [8e10]. Indeed, experiments performed in vivo and in vitro have highlighted the positive effects of GH and IGF-I on uterine functions, increasing mRNA expression for proteins that form the histotroph and on conceptus development during the pre- and peri-implantation stages in many species [11e15]. Recombinant bovine somatotropin (bST) is commonly utilized in dairy operations to improve the milk production of lactating dairy cows [16]. The administration of bST increases plasma concentrations of IGF-1 in both beef and dairy cattle [17e19]. However, the literature presents contradictory results regarding the effect of bST on improving fertility, specifically P/AI [17,20]. Ribeiro et al. [21] reported that low doses of bST during the pre- and peri-implantation periods enhanced conceptus development, reduced embryonic losses and improved fertility in dairy cows. They also reported an increase of IGF-I for two weeks after applying 325 mg of bST on D0, and for four weeks when applying it on D0 and D14; however, such doses were not sufficient to alter the plasma progesterone concentrations. Also, Moreira et al. [22] administered 500 mg of bST to embryo donors and/or recipients and found that the administration of bST increased the percentage of transferable embryos, the number of blastocysts obtained by uterine flush, and pregnancy per embryo transfer. In contrast, Rivera et al. [20], using primiparous and multiparous Holstein cows, studied the effect of 6 consecutive subcutaneous injections of 500 mg of bST given at 10-d intervals, starting around 60 days postpartum. They concluded that this bST treatment improved lactation performance but had no effect on P/AI. In beef cattle, some researchers have evaluated the effect of bST on P/AI using different doses and times of application. Starbuck et al. [23] administered 500 mg bST at the time of AI on lactating dairy cows, dairy heifers and lactating beef cows; they found an increase in conception rates only in dairy cows. Mercadante et al. [19] administered a dose of 325 mg of bST at TAI, 14 days after TAI or on both days to multiparous, suckled beef cows. They found an increase in IGF1 plasmatic concentration; however, they did not report any improvement in P/AI, fetal size or plasma progesterone. Oosthuizen et al. [24] used 650 mg of bST at the moment of CIDR insertion (9 days before TAI) in crossbred beef heifers. They reported greater P/AI in the control group (no bST) compared to bST heifers. Cooke et al. [25] evaluated the effect of 250 mg of bST in pubertal beef heifers on plasmatic glucose, insulin, IGF1 and progesterone concentration. They found that bST failed to affect the concentrations of insulin and P4, but increased those of glucose and IGF1. In this context, it was hypothesized that treatment with bST on the day of FTAI or 7 days later would increase P/AI in Bos indicus cows compared with untreated controls. It has also been hypothesized that bST could promote an increase in the size of the

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endometrial glands, but not in the size of the corpus luteum (CL) or plasma progesterone concentrations. In order to test our hypotheses, three experiments were designed. The aim of Experiment 1 was designed to evaluate pregnancy per artificial insemination (P/AI) in cows treated with 0, 250 or 500 mg of bST on the day of the FTAI (Day 0). In Experiment 2, the aim was to evaluate the effect of administering 500 mg of bST 7 days after the FTAI (Day 7). Finally, in Experiment 3, the aim was to evaluate the effect of 500 mg bST, administered on the ovulation day (Day 0), on the cellular composition of the CL and the morphometry of the endometrial glands 15 days after administration (Day 15) in heifers. 2. Materials and methods 2.1. Experiment 1 2.1.1. Animals and management All experimental procedures were approved by the Ethics ~o Paulo State UniverCommittee on Animal Experimentation of Sa ~o Paulo, Brazil (Protocol number sity (UNESP), Araçatuba, state of Sa 2013/00602). The experiment was conducted in Araguaiana, state of Mato Grosso, Brazil. Multiparous Nelore cows (Bos taurus indicus; n ¼ 587) between 40 and 70 days postpartum were used in this experiment. The means and standard deviation (SD) for body condition score (scale 1e5, where 1 ¼ emaciated and 5 ¼ overconditioned) were 3.01 þ 0.21 for all treatments. The cows were kept on Brachiaria brizantha pastures with water and mineral supplementation ad libitum. The experiment was conducted using a randomized block design: first cows were randomly divided into eight lots for management purposes and all were submitted to FTAI on the same day. Then, within each lot, cows were assigned randomly to the bST 0, bST 250 or bST 500 treatments. 2.1.2. Synchronization of ovulation Intravaginal devices containing 1 g of progesterone (DIB®; MSD ~o Paulo, SP, Brazil) were used, associated with the Animal Health, Sa intramuscular (IM) injection of 2 mg of estradiol benzoate (Gonadiol®, 1 mg/mL; MSD Animal Health) on the day of the device insertion. The devices were removed after 8 days, at which time the cows were treated with 0.530 mg of sodium D-cloprostenol (Ciosin®, 0.265 mg/mL; MSD Animal Health) and 300 IU of equine chorionic gonadotropin (eCG; Novormon®, 200 IU/mL; MSD Animal Health). Cows were treated with 1 mg of estradiol benzoate (Gonadiol®; MSD Animal Health) IM 24 h after removal of the devices; 30 h after the last injection, the cows were submitted to FTAI. Ovaries were examined using ultrasonography (Aloka Ultrasound Diagnostic Equipment, Model SSD-500, Tokyo, Japan) on the day of the progesterone device placement and 30% of the cows presented a visible CL. 2.1.3. Fixed timed artificial insemination procedure AI was performed using frozen semen straws containing sperm from 3 Aberdeen Angus bulls. Inseminations were performed by three inseminators, equally divided between the different treatments. 2.1.4. Treatment with bST During the FTAI procedure, cows were randomly assigned into one of the experimental treatments. The cows received 0 mg (bST 0; n ¼ 194), 250 mg (bST 250; n ¼ 197) or 500 mg (bST 500; n ¼ 196) of bST (Boostin®, 250 mg/mL; MSD Animal Health), administered subcutaneously (SC) in the ischiorectal fossa. 2.1.5. Pregnancy diagnosis Pregnancy diagnosis was performed 30 days after FTAI (D30)

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using ultrasonography (Aloka Ultrasound Diagnostic Equipment, Model SSD-500, Tokyo, Japan) with a 5 mHz linear transducer. Females were classified as pregnant (presence of an amniotic vesicle containing a live embryo) or not pregnant. 2.1.6. Statistical analysis Data were analyzed by logistic regression (GLIMMIX procedure of SAS Institute, Cary, NC, USA). The initial model for the analysis of P/AI included the effects of treatment, lot, body condition score and interactions. In order to obtain the final statistical model, the explanatory variables were sequentially removed according to the Wald statistic criterion, removing variables with P > 0.20; in the end, only the effect of treatment remained in the model. In the analysis, P < 0.05 was considered significant. 2.2. Experiment 2 2.2.1. Animals and management All experimental procedures were approved by the Ethics Committee on Animal Experimentation of the UNESP, Dracena, S~ ao Paulo state, Brazil (Protocol number 02/2008). The experiment was ~o Fe
lix do Araguaia, Mato Grosso state, Brazil. conducted in Sa Multiparous Nelore cows (Bos taurus indicus; n ¼ 243) between 35 and 70 days postpartum were used. The means and SD for body condition score (scale 1e5, where 1 ¼ emaciated and 5 ¼ overconditioned) were 2.82 ± 0.23 and 2.9 ± 0.02 for the bST 0 and bST 500 treatments, respectively. The females were kept on Brachiaria brizantha and Panicum maximum pastures with water and mineral supplementation ad libitum. 2.2.2. Synchronization of ovulation Intravaginal devices containing 1 g of progesterone (DIB®; MSD Animal Health) were used, associated with an IM injection of 2 mg of estradiol benzoate (Gonadiol®; MSD Animal Health) on the day of the placement of the devices. The devices were removed after 8 days, when the cows were treated with 112.5 mg of D-cloprostenol (Preloban®; 0.075 mg/Ml; Intervet Schering-Plough, S~ ao Paulo, Brazil) and 300 IU of eCG (Folligon®; 200 IU/Ml; Intervet ScheringPlough), IM. Cows were treated with 1 mg of estradiol benzoate IM (Gonadiol®; MSD Animal Health) 24 h after removal of the devices and were submitted to FTAI 30 h after the last injection. 2.2.3. FTAI procedure AI was performed using the semen of 14 bulls. Each bull was distributed equally among all the treatments and randomly between the cows. Inseminations were performed by two farm technicians, who were also distributed equally among all the treatments. 2.2.4. Treatment with bST Seven days after FTAI (D7), cows were randomly distributed to receive 0 mg (bST 0; n ¼ 124) or bST (Boostin®; MSD Animal Health) at a dose of 500 mg (bST 500; n ¼ 119), SC in the ischiorectal fossa. 2.2.5. Collection of blood samples A random selection of 39.5% (96/243) of the cows was used to perform progesterone concentration analyses. Blood collections were performed on D7 and D16 after FTAI. Approximately 4 mL of blood was collected with a vacutainer (Becton, Dickinson and Company; BD) containing 175 mL of 30% sodium citrate by venipuncture of the median caudal vein or artery; these were stored at 4  C. The samples were centrifuged at 2900g for 30 min at room temperature (Centribio Centrifuge, Model 80-2B). After centrifugation, the plasma was removed, put into microtubes and stored

at 20  C. 2.2.6. Radioimmunoassay for the measurement of progesterone The progesterone concentrations were measured by radioimmunoassay using commercial kits (Count-a-Count®; DPC, Diagnostic Products Corporation, Los Angeles, USA). Samples were measured in three runs; samples containing standard high concentrations (HCS) and low concentrations (LCS) of progesterone were used. For HCS, the intra-assay coefficients of variation were 1.60, 1.56 and 1.39% for runs 1, 2 and 3, respectively. For LCS, the intra-assay coefficients of variation were 0.15, 0.19 and 0.11% for runs 1, 2 and 3, respectively. The inter-assay coefficients of variation were 5.30% for HCS and 4.54% for LCS. The sensitivity limits for runs 1, 2 and 3 were 0.058, 0.054 and 0.158 ng/mL, respectively. 2.2.7. Pregnancy diagnosis Pregnancy diagnosis was performed 30 days after FTAI (D30) using ultrasonography (Aloka Ultrasound Diagnostic Equipment, Model SSD-500, Tokyo, Japan) with a 5 mHz linear transducer. Females were classified as pregnant (presence of an amniotic vesicle containing a live embryo) or not pregnant. 2.2.8. Statistical analysis Statistical analysis was performed using SAS statistical software. The initial model for the analysis of P/AI included the effects of treatment, lot, body condition score and interactions. In order to obtain the final statistical model, the explanatory variables were sequentially removed according to the Wald statistic criterion, removing variables with P > 0.20; in the end, only the effect of treatment remained in the model. In the analysis, P < 0.05 was considered significant. Using the PROC MIXED procedure of SAS, ANOVA analyses were used to compare the progesterone concentrations on D7 and D16 between the 0 and 500 mg of bST groups. Therefore, progesterone concentrations on D7 were used as the covariate in the statistical analysis of progesterone on Day 16. The residuals were checked for heteroscedasticity, which proved adequate. The significance level used for all data was P  0.05. All results were presented as means ± standard errors of the mean (SEM). 2.3. Experiment 3 2.3.1. Animals and management All experimental procedures were approved by the Ethics Committee on Animal Experimentation of the UNESP, Araçatuba/SP, Brazil (Protocol number 2013/00602). The experiment was con~o Paulo, Brazil. Non-pregnant, cyclic ducted in Birigui, state of Sa nulliparous Nelore heifers (Bos taurus indicus; n ¼ 24) were used. Of the 24 heifers, 20 ovulated and remained in the study. Females were previously evaluated for age, weight and body condition score. The means and SD for body condition score (scale 1e5, where 1 ¼ emaciated and 5 ¼ over-conditioned) were 2.87 ± 0.31 for both treatments. Ovarian condition was evaluated by ultrasonography (Aloka Ultrasound Diagnostic Equipment, Model SSD-500, Tokyo, Japan) on the day of placement of the devices, when 40% of females presented a CL. The females were kept in confinement, receiving a diet containing 40.7% sugar cane, 28.3% corn germ, 16.7% orange pulp, 11.3% cottonseed meal, 1% urea and 2% mineral core. 2.3.2. Synchronization of ovulation Intravaginal devices containing 1 g of progesterone (DIB®; MSD Animal Health) were used, associated with an IM injection of 2 mg of estradiol benzoate (Gonadiol®; MSD Animal Health) on the day of placement of the devices. The devices were removed after 8 days, when cows were treated with 0.530 mg of D-cloprostenol (Ciosin®;

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MSD Animal Health) and 300 IU of eCG (Novormon®; MSD Animal Health). The heifers were treated with 1 mg of estradiol benzoate (Gonadiol®; MSD Animal Health) intramuscularly 24 h after the removal of the devices. Considering that ovulation occurs 36e40 h after the administration of estradiol benzoate, D0 was determined to be the day of ovulation. Ovulation was confirmed by ultrasonography 48 h after the application of estradiol benzoate. The females were not inseminated.

and small diameters of each cell were individually measured, and the mean between both measurements was taken in order to rank each cell. An arbitrary size cutoff of 20 mm was used for separating SLC from LLC [26,27]. To estimate the percentages of SLC and LLC, 200 cells were counted in 16 slides/animal and a single mean per cow was calculated; this evaluation was performed automatically using Windows Select Measurements by measuring randomly selected fields.

2.3.3. Treatment with bST Thirty hours after the last injection of estradiol benzoate (D0 ¼ ovulation day), heifers received 0 mg (bST 0; n ¼ 9) or bST (Boostin®; MSD Animal Health) at a dose of 500 mg (bST 500; n ¼ 11), applied SC in the ischiorectal fossa. The heifers were slaughtered on D15 (D0 ¼ ovulation day).

2.3.9. Morphometric analysis of endometrial glands After obtaining the histology slides, they were subjected to computerized image analysis programmed in a light microscope with a camera (ZEISS Axioplan 2, Model: MC 80 DX - Microscope Camera). Photos of 10 randomly selected optical fields were taken, five of which were from the endometrial superficial region and five from the deep region, with a 10x magnifying lens. Afterward, the photos were analyzed using the software Image-Pro® PLUS-The Proven Solution™ to calculate the number, perimeter (mm) and area (mm2) of ducts of the endometrial glands in the superficial endometrium and the deep endometrium.

2.3.4. Collection of blood samples Blood collections were performed immediately after slaughter. Approximately 4 mL of blood was collected with a vacutainer (Becton, Dickinson and Company; BD) containing 175 mL of 30% sodium citrate by venipuncture of the median caudal vein or artery; these were stored at 4  C. The samples were subjected to centrifugation at 2900g for 30 min at room temperature (Centribio Centrifuge, Model 80-2B). After centrifugation, the plasma was removed, put into microtubes and stored at 20  C. 2.3.5. Radioimmunoassay for the measurement of progesterone The progesterone concentrations were measured by radioimmunoassay using commercial kits (Count-a-Count®; DPC, Diagnostic Products Corporation, Los Angeles, USA). All samples were analyzed in a single run. Samples containing standard high concentrations (HCS) and low concentrations (LCS) of progesterone were used. For HCS and LCS, the intra-assay coefficient of variation was 1.49% and 1.62%, respectively. The sensitivity limit for the assay was 0.15 ng/mL.

2.3.10. Statistical analysis Statistical analysis was performed using SAS statistical software. The residuals were checked for heteroscedasticity, which proved adequate; only the progesterone concentration data were transformed using a natural logarithm. Using the GLM procedure, the effect of the treatment on the dependent variables was evaluated with an ANOVA. The treatment effect (0 or bST 500 treatment) was evaluated, considering cow as a random effect and cow within each treatment. The cows were considered as an experimental unit. In all analyses, a level of P  0.05 was considered significant. All results were presented as means ± standard errors of the mean (SEM). 3. Results 3.1. Experiment 1

2.3.6. Collection of luteal and uterine tissue Immediately after slaughter, the ovaries were removed, weighed and their diameters were measured separately. The CL was isolated, weighed and its diameter was measured. A segment of the uterine horn was removed in the mid-third of the horn, ipsilateral to the CL. Both tissues were immediately packaged in buffered 4% formalin (pH 7.0) for 24 h. The fragments were washed six consecutive times every 12 h with PBS solution pH 7.5. 2.3.7. Histological preparation of tissues The tissues were dehydrated by immersion in 70% alcohol for 60 min, then 90% alcohol for 60 min, followed by two 60-min washes in absolute alcohol, and, finally, washed twice for 60 min in xylene. The tissue was ‘embedded’ in paraffin. The CL was cut into 6- to 8-mm-thick cross sections and the uterine segment was cut into 4-mm-thick endometrial sections. Slides were placed in the incubator for 2 h at temperatures of 56e58  C, then stained with hematoxylin-eosin (HE). 2.3.8. Assessment of steroidogenic luteal cells The stained slides were subjected to a computerized image analysis system (Leco 2001, MI, USA), coupled with a camera (ZEISS Axioplan 2, Model: MC 80 DX - Microscope Camera, Zeiss, Oberkochen, Germany). Using a light microscope, size, shape and morphology were used to distinguish luteal cells from non-luteal cells, as well as small luteal cells (SLC) from large luteal cells (LLC). LLC appear polyhedral in shape and contain lipid droplets, whereas SLC were stained darker than LLC cells. The cell diameter was determined by individual distance measurements. The large

As shown in Table 1, pregnancy per artificial insemination did not differ between treatments (P ¼ 0.26). These values were 59.28% (115/194), 58.38% (115/197) and 65.82% (129/196) for the bST 0, bST 250 and bST 500 treatments, respectively. 3.2. Experiment 2 As shown in Table 1, pregnancy per artificial insemination did not differ between treatments (P ¼ 0.59) and was 57.3% (71/124) and 60.5% (62/119) for the bST 0 and bST 500 treatments, respectively. Concentrations of progesterone in plasma on D7, before

Table 1 Pregnancy per AI (P/AI) in Nelore cows subjected to fixed timed artificial insemination and treated with either 0, 250 or 500 mg of bovine somatotropin on Day 0 at AI (Experiment 1) or treated with 0 or 500 mg of bovine somatotropin on Day 7 (Experiment 2). Variable

Pregnancy per AI (P/AI)

Experiment 1 bST 0 bST 250 bST 500

59.28% (115/194) 58.38% (115/197) 65.82% (129/196)

P value

P ¼ 0.26 Experiment 2 bST 0 bST 500

57.3% (71/124) 60.5% (62/119) P ¼ 0.59

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treatments were applied, did not differ between bST 0 or bST 500 (4.73 ± 0.33 ng/mL vs. 5.48 ± 0.38 ng/mL) treatments. Plasma progesterone concentrations on D16 were smaller than 1.0 ng/mL in 5 out of 96 cows evaluated (4 from bST 0 and 1 from bST 500 group), indicating that the cows underwent luteolysis by D16. Therefore, when considering only those cows with an active CL based on concentration of progesterone in plasma greater than 1.0 ng/mL on D16 were again greater (P ¼ 0.02) in the bST 500 treatment (11.63 ± 0.84 ng/mL) than in the bST 0 treatment (9.83 ± 0.88 ng/ mL). 3.3. Experiment 3 As shown in Table 2, there were no differences between the bST 0 and the bST 500 treatments for the diameter and weight of the ovary, diameter on the CL, and concentration of progesterone in plasma. The bST 500 group had a heavier CL compared to the bST 0 treatment (P ¼ 0.04). There was no difference between the bST 0 and the bST 500 treatment for the percentage of SLC (86.28 ± 1.39% vs. 91.40 ± 1.52%, respectively; P ¼ 0.07). However, the percentage of LLC was lower in the bST 500 treatment than in the bST 0 treatment (8.60 ± 1.52 vs. 13.72 ± 1.16%, respectively; P ¼ 0.01). There were no significant differences between the bST 0 and bST 500 treatments in terms of the number, perimeter and area of the superficial endometrial glands, deep endometrial glands and the total endometrial glands. 4. Discussion The present experiment evaluated the effects of a single dose of bST during FTAI protocols on beef cattle. In experiments 1 and 2, the P/AI did not differ between treatments. The use of bST in FTAI protocols has contradictory results in the literature. Albuquerque et al. [28] used a similar synchronization protocol in Nelore cows to the one we used here, with an additional administration of 167 and 333 mg of bST on the day of the FTAI. These same authors found P/ AI of 37.4% in the 0 mg treatment, 45.4% in the 167 mg treatment, and 46% in the 333 mg treatment; they found a significant treatment effect but not a dose effect. In a second experiment, the same authors used two administrations of 333 mg of bST the day of FTAI and 11 days after. The P/AI was 48.0% in the control and 42.3% in the treatment. This could indicate that bST improve the P/AI only when the control group has very low results, meaning cows with low BCS or nutritional deficiencies. In the current study, the bST 0 group

presented higher pregnancy per artificial insemination than those reported for commercial herds, which leads to the assumption that cows with low body condition scores may possibly be favored in the use of bST. In several studies, the use of bST was found to promote a gradual increase in the plasma concentrations of GH and IGF-I during the first 7 days, when the peak of these hormones is observed, with a continued daily gradual decline [21,29]. Thus, it is assumed that the application of bST at the time of artificial insemination promotes a sustained increased in concentrations of GH and IGF-I during the period coinciding with the beginning of embryonic development through the seventh day. Kolle et al. [8] demonstrated that GH receptors are present in the embryo beginning from the two-cell stage, when it acts on glycogen metabolism and the transport of lipids to the embryo. Spencer et al. [12] showed that the glandular epithelium of the ovine uterus responded to the intrauterine administration of GH by increasing the mRNA expression of the proteins that constitute the histotroph; this condition favored a higher production of IFN-t. However, when the administration of bST is performed 7 days after FTAI, it is assumed that the greatest effects would occur from the 7th to the 14th days of the development of the conceptus, which would be the elongation stage. Ribeiro et al. [21] observed that treating dairy cows with 350 mg of bST on the day of artificial insemination did not alter the size of the conceptus or fertility; however, administration of 350 mg of bST on the day of AI and 14 days later increased the development of the conceptus, reduced embryo mortality and increased fertility. The authors found that the use of two doses increased the longitudinal diameter of the amniotic vesicle and the size of the fetus at 34 and 48 days of pregnancy; however, there was no effect on the P/AI. The increase of cellular proliferation and elongation of the conceptus are extremely important in order to prevent luteolysis and maintain pregnancy [12]. Such associated events potentiate embryo development, increasing the chances of embryo survival. Considering these reports, one would expect an increase in P/AI in the present experiment; however, this did not hold true. Rossetti et al. [30] also found that the application of 500 mg of bST 7 days after FTAI did not increase the conception P/AI rates in Nelore cows, which were 57.3% in placebo and 61.1% in the bST group. Even when bST was associated with the application of flunixin meglumine and/ or human chorionic gonadotropin (hCG), 16 days after the FTAI, there was no increase in fertility or the concentration of P4. In Experiment 3, the use of a single dose of 500 mg of bST on D0 failed to promote changes in ovaries size and weight; however, bST

Table 2 Experiment 3: Means ± standard errors of the mean (SEM) of ovarian and luteal diameter and weight, luteal and endometrial microscopic characteristics and progesterone concentrations in heifers who received 30 h after the last injection of estradiol benzoate, 0 or 500 mg bST, applied SC in the ischiorectal fossa, slaughtered on D15 (D0 ¼ ovulation day). Variable

bST 0 (n ¼ 9)

bST 500 (n ¼ 11)

P value

Ovarian diameter (cm) Ovarian weight (g) Luteal diameter (cm) Luteal weight (g) Large luteal cells (% of total luteal cells) Small luteal cells (% of total luteal cells) Number of superficial gland ducts Mean superficial gland duct perimeter (mm) Mean superficial gland duct area (mm2) Number of deep gland ducts Mean deep gland duct perimeter (mm) Mean deep gland duct area (mm2) Total gland duct density Mean total gland duct perimeter (mm) Mean total gland duct area (mm2) Progesterone concentrations (ng/mL)

2.06 ± 0.13 5.61 ± 0.66 1.33 ± 0.06 2.25 ± 0.20 13.72 ± 1.16 86.28 ± 1.39 16.46 ± 3.14 247.66 ± 18.23 3955.52 ± 487.68 28.75 ± 3.25 189.25 ± 7.53 2110.30 ± 114.14 46.22 ± 5.86 456.96 ± 28.04 6571. 59 ± 765.12 11.06 ± 1.43

2.27 ± 0.09 6.50 ± 0.48 1.54 ± 0.08 3.11 ± 0.32 8.60 ± 1.52 91.40 ± 1.52 17.34 ± 2.03 251.53 ± 17.34 3885.93 ± 476.58 29.94 ± 1.69 198.82 ± 10.83 2153.33 ± 196.11 47.25 ± 2.64 453.05 ± 19.63 6052.43 ± 555.09 9.92 ± 1.43

0.21 0.28 0.06 0.04 0.01 0.07 0.97 0.88 0.92 0.74 0.50 0.86 0.86 0.97 0.58 0.58

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led to an increase in the weight of the CL and plasma progesterone concentrations on D16 were greater in the bST 500 treatment than in the bST 0 treatment (11.63 ± 0.84 ng/mL vs. 9.83 ± 0.88 ng/mL). As noted in the present study, some in vivo and in vitro studies have shown that bST favors the growth of luteal tissue. GH receptors (transcripts and proteins) have been previously found in the CL and LLC [31]. The administration of bST also has been shown to increase the weight of the CL [31], but it did not increase the plasma concentrations of P4 in dairy cows [21]. Lucy et al. [31] treated cows with 25 mg of bST daily for 16 days after estrus and then, on day 17, cows were slaughtered to collect ovaries, uterus and the conceptus; bST increased CL weight and the number of large follicles (10e15 mm in diameter). In this study, note that differences in ovulatory follicle diameter may have resulted in differences in CL size and weight, as a function of the number of granulosa and theca cells that were luteinized, which would not be related to treatments. Later studies, however, have failed to show a positive effect of bST on the CL during the peri-ovulatory period [31]. In the present study, bST led to an increase in the weight of the CL and a decrease in the percentage of LLC in heifers. Hence, bST altered the cellular composition of the CL, but not the concentration of P4. In fact, experiments that showed improvements in P/AI with bST did not associate the positive effects on fertility to be mediated by increased concentrations of P4 [21]. We conclude that the doses of bST used in this study do not promote increased P/AI, although bST altered the cellular composition of the CL and increased the P4 concentration in the late diestrus. Acknowledgements The authors wish to acknowledge the Coordination for the Improvement of Higher Education Personnel (CAPES), the Development Foundation of UNESP (FUNDUNESP) and MSD Animal Health. References [1] Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGF2alpha and GnRH. Theriogenology 1995;44(7):915e23. [2] Baruselli PS, Sales JNS, Sala RV, Vieira LM, Sa Filho MF. History, evolution and perspectives of timed artificial insemination programs in Brazil. Anim Reprod 2012;9(3):139e52. [3] Meneghetti M, Sa Filho OG, Peres RFG, Lamb GC, Vasconcelos JLM. Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows I: basis for development of protocols. Theriogenology 2009;72(2):179e89. [4] Santos JE, Thatcher WW, Chebel RC, Cerri RL, Galvao KN. The effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs. Anim Reprod Sci 2004;82e83:513e35. [5] Breuel KF, Lewis PE, Schrick FN, Lishman AW, Inskeep EK, Butcher RL. Factors affecting fertility in the postpartum cow: role of the oocyte and follicle in conception rate. Biol Reprod 1993;48(3):655e61. [6] Binelli M, Thatcher WW, Mattos R, Baruselli PS. Antiluteolytic strategies to improve fertility in cattle. Theriogenology 2001;56(9):1451e63. [7] Hansen P. Embryonic mortality in cattle from the embryo's perspective. J Anim Sci 2002;80(E-Suppl 2):E33e44. [8] Kolle S, Stojkovic M, Prelle K, Waters M, Wolf E, Sinowatz F. Growth hormone (GH)/GH receptor expression and GH-mediated effects during early bovine embryogenesis. Biol Reprod 2001;64(6):1826e34. [9] Yaseen MA, Wrenzycki C, Herrmann D, Carnwath JW, Niemann H. Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems. Reproduction

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