Associations of antral follicle count with fertility in cattle: A review.

Ovarian antral follicle count (AFC) is a marker of ovarian stimulatory response to superovulation protocols in cattle. This article reviews novel research from the past 10 years, focusing on the relationship between AFC and embryo production and cow fertility. Substantial evidence indicates a positive relationship between AFC with embryo production; however, conflicting findings exist regarding the relationship of AFC with conception and pregnancy rates. This lack of consistent association with pregnancy outcomes is perplexing given the differences detected in oocytes, embryos, and endometria from high- versus low-AFC animals. Those differences include markers of embryonic viability such as protein level, blastocyst development rates, cleavage rates, and blastocyst cell numbers that differ between high- and low-AFC groups, as well as differential gene expression at the cow and embryo level with genes associated with fertility. In addition, Bos indicus and Bos taurus cattle appear to have different fertility responses based on their AFC category. In summary, clearly more studies are needed to elucidate the true associations between AFC and cow fertility, but the data that have been accumulated thus far indicate that AFC has the potential to be a useful marker of lifetime cow fertility.

Comparison of production-related responses to hyperinsulinemia and hypoglycemia induced by clamp procedures or heat stress of lactating dairy cattle.

Hyperinsulinemia concurrent with hypoglycemia is one of a myriad of physiological changes typically experienced by lactating dairy cows exposed to heat stress, the consequences of which are not yet well defined or understood. Therefore, the objective of this experiment was to separate the production-related effects of hyperinsulinemia with hypoglycemia from those of a hyperthermic environment. Multiparous lactating Holstein cows (n = 23; 58 ± 4 d in milk, 3.1 ± 0.3 lactations) were housed in temperature-controlled rooms and all were subjected to 4 experimental periods as follows: (1) thermoneutral (TN; temperature-humidity index of 65.1 ± 0.2; d 1-5), (2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; insulin infused at 0.3 µg/kg of BW per h, glucose infused to maintain 90 ± 10% of baseline blood glucose for 96 h; d 6-10), (3) heat stress (HS; temperature-humidity index of 72.5 ± 0.2; d 16-20), and (4) HS + euglycemic clamp (EC; glucose infused to reach 100 ± 10% of TN baseline blood glucose for 96 h; d 21-25). Cows were fed and milked twice daily. Feed refusals were collected once daily for calculation of daily dry matter intake, and milk samples were collected at the beginning and end of each period for component analyses. Circulating insulin concentrations were measured in daily blood samples, whereas glucose concentrations were measured more frequently and variably in association with clamp procedures. Rectal temperatures and respiration rates were greater during HS than TN, as expected, and states of hyperinsulinemia and hypoglycemia were successfully induced by the HHC and high ambient temperatures (HS and EC). Feed intake differed based upon thermal environment as it was similar during TN and HHC periods, and declined for HS and EC. Milk production was not entirely reflective of feed intake as it was greatest during TN, intermediate during HHC, and lowest during HS and EC. All milk components differed with the experimental period, primarily in response to the thermal environment. Interestingly, TN baseline glucose concentrations were highly correlated with the change in glucose from TN to HS, and were related to glycemic status during HS. Furthermore, although few in number, those cows that failed to become hypoglycemic during HS tended to have a greater reduction in milk yield. The work presented here addresses a critical knowledge gap by broadening our understanding of the physiological response to heat stress and the related changes in glycemic state. This broadened understanding is fundamental for the development of novel, innovative management strategies as the dairy industry is compelled to become increasingly efficient in spite of global warming.

Effects of progesterone concentrations and follicular wave during growth of the ovulatory follicle on conceptus and endometrial transcriptome in dairy cows.

Objectives were to evaluate the effects of follicular wave and progesterone concentration on growth of the ovulatory follicle, conceptus elongation, uterine IFN-τ concentration, and transcriptome of conceptus and endometrium of pregnant cows on d 17 of gestation. Nonlactating nonpregnant Holstein cows were assigned randomly to one of 3 treatments: ovulation of a first-wave follicle (FW, n = 15); ovulation of a first-wave follicle and progesterone supplementation (FWP4, n = 12); and ovulation of a second-wave follicle (SW, n = 19). Ovulation of a first- or second-wave follicle was achieved by initiating the Ovsynch protocol (d -9 GnRH, d -2 and -1 PGF2α, d 0 GnRH and artificial insemination, d 0.7 artificial insemination) on d 0 or 6 of a presynchronized estrous cycle, respectively. Cows in FWP4 received 3 intravaginal inserts containing progesterone at 12, 24, and 48 h after the first GnRH injection that were removed on d -2. Cows were killed on d 17 for collection of the reproductive tract. Transcriptome was evaluated by microarray using the Affymetrix Bovine Array. Orthogonal contrasts were built to assess the effects of progesterone concentration during follicle growth (FW vs. FWP4 + SW) and follicular wave (FWP4 vs. SW). Progesterone concentrations (LSM ± SEM) from d -9 to -2 were greater for SW, followed by FWP4 and FW (5.38 ± 0.24, 4.26 ± 0.28, and 1.17 ± 0.27 ng/mL). Diameter of the ovulatory follicle (FW = 19.6 ± 0.6; FWP4 = 15.6 ± 0.6; SW = 15.2 ± 0.5 mm) and concentrations of estradiol from d -2 to 1 (FW = 4.05 ± 0.33; FWP4 = 2.73 ± 0.35; SW = 2.48 ± 0.30 pg/mL) were greater for FW compared with FWP4 and SW. Progesterone concentrations from d 3 to 16 were greater for FW compared with FWP4 and SW. A total of 28 singleton conceptuses were collected (FW, n = 8; FWP4, n = 8; SW, n = 12) and only intact conceptuses were included in the analyses of length (FW, n = 8; FWP4, n = 6; SW, n = 12). Although conceptuses were longer for FW compared with FWP4 and SW (FW = 16.6 ± 2.3; FWP4 = 9.8 ± 2.2; SW = 9.6 ± 2.0 cm), treatment did not affect the amount of IFN-τ in uterine flushing. Transcriptome of conceptuses and endometrium of pregnant cows was not extensively affected by follicular wave (8 and 1 differentially expressed transcripts) or concentration of progesterone during follicle growth (0 and 3 differentially expressed transcripts), showing that these factors did not affect conceptuses and endometrium transcriptome in pregnancies that are maintained to d 17.

Effects of nutrient restriction on the metabolic profile of Bos indicus-influenced and B. taurus suckled beef cows.

Recent research from our group demonstrated that Bos indicus-influenced suckled beef cows had greater resilience to withstand nutrient restriction and establish pregnancy compared with B. taurus cows exposed to the same conditions. To further understand these findings, differences in metabolic profile between these same B. indicus-influenced and B. taurus females were explored. Suckled beef cows (n = 134) were enrolled in a completely randomized design with a 2 × 2 factorial arrangement of treatments. On day -21, Angus (AN; Bos taurus) and Brangus (BN; B. indicus-influenced) cows were randomly assigned to 1) a diet that met daily energy maintenance requirements (MAINT), or 2) a diet that restricted intake to 70% of the daily energy maintenance requirements (RESTR). Cows were exposed to an estrus synchronization protocol and received an embryo 7 d after ovulation was pharmacologically induced on day 0. Blood samples were collected on days -21 and 19 to determine circulating concentrations of non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), insulin, glucose, and IGF-1. Pregnancy status after embryo transfer was determined on day 28. As a consequence of the proposed diets, cows in the RESTR diet had less body condition score (BCS) on day 19 (P = 0.008) across breed types. Moreover, BCS change from day -21 to 19 was included as independent covariate into subsequent analyses, allowing for the comparison of breed types under an equivalent level of body reserve mobilization. A breed × diet interaction was observed for plasma insulin (P = 0.03) and IGF-1 (P = 0.04) on day 19, where AN-RESTR cows had less plasma concentrations on day 19 compared with AN-MAINT cows. Diets did not impact (P > 0.10) plasma insulin and IGF-1 concentrations in BN cows. No diet or breed effects were observed in circulating concentrations of NEFA, BHB, and glucose (P > 0.10). Across breed types and nutritional treatment, there was positive linear effect (P ≤ 0.04) of plasma concentrations of insulin and IGF-1 on the probability of pregnancy to fixed-time embryo transfer. In summary, the negative impacts of nutrient restriction on the somatotropic axis, independently of body tissue mobilization, were heightened in Bos taurus females compared with B. indicus-influenced cohorts, which corroborate with the differences observed in fertility between these subspecies.

Graduate Student Literature Review: Potential mechanisms of interaction between bacteria and the reproductive tract of dairy cattle.

Although the presence of bacteria has been characterized throughout the reproductive tracts of multiple species, how these bacteria may interact with the host has yet to be described. Previous reviews have described how pathogenic bacteria interact with the reproductive tract to cause infections such as metritis. This review aimed to summarize the knowledge related to pathogenic and nonpathogenic bacteria in various locations of the bovine reproductive tract and the possible mechanisms underlying host-microbe interactions during gametogenesis and early pregnancy. Lactic acid bacteria such as Lactobacillus seem to be beneficial in multiple areas of the reproductive tract: they have been associated with increased oocyte quality when in follicular fluid and secrete reactive oxygen species that are beneficial during placental angiogenesis. However, other bacteria, including Enterococcus, Staphylococcus, and Streptococcus, may modulate T helper cells that inhibit maternal recognition of pregnancy. Available data on the reproductive microbiome focus on variations in microbial communities and their associations with reproductive performance. However, research on these host-microbiome interactions may provide more insight on how bacteria affect fertility.

Propionate Affects Insulin Signaling and Progesterone Profiles in Dairy Heifers.

Emerging data highlighting gut microbiome influences on health support evaluation of how microbial fermentation end-products influence postabsorptive systems. This study aimed to investigate the effect of increased propionate status on progesterone profiles and insulin sensitivity in dairy heifers. Eleven Holstein heifers, synchronized in estrus, were assigned to one of two continuous, 5-day IV treatments: sodium propionate (PRO; n = 5) or saline (CON; n = 6). These infusions culminated in a hyperglycemic clamp with daily blood samples for an additional 7 days. Plasma propionate concentrations increased over the first 9 h in PRO heifers, then decreased until day 3 when they matched CON heifers. Maximum plasma progesterone concentrations tended to be greater in PRO heifers than CON heifers (4.19 vs 3.73 ng/mL; P = 0.087). Plateau insulin concentrations in CON animals were significantly greater than those in PRO animals (249.4 ± 25.1 vs 123.9 ± 35.8; P = 0.008) with a trend for an increased insulin sensitivity index in PRO heifers compared to CON heifers (P = 0.06). These changes in plasma propionate clearance leading to increased progesterone response and changes in insulin sensitivity suggest a role for SCFA metabolism in reproductive hormone regulation.

Activities for leptin in bovine trophoblast cells.

Leptin is involved in various reproductive processes in humans and rodents, including placental development and function. The specific ways that leptin influences placental development and function in cattle are poorly understood. This work was completed to explore how leptin regulates hormone, cytokine and metalloprotease transcript abundance, and cell proliferation in cultured bovine trophoblast cells. In the first set of studies, cells were cultured in the presence of graded recombinant bovine leptin concentrations (0, 10, 50, 250 ng/mL) for 6 or 24 h. Transcript profiles were examined from extracted RNA. Leptin supplementation did not affect abundance of the maternal recognition of pregnancy factor, interferon-tau (IFNT), but leptin increased (P < 0.05) abundance of chorionic somatomammotropin hormone 2 (CSH2; ie, placental lactogen) at both 6 and 24 h at each concentration tested. At 24 h, the greatest CSH2 abundance (P < 0.05) was detected in cells supplemented with 50 ng/mL leptin. Transcript abundance of the remodeling factor, metalloprotease 2 (MMP2), was greater (P < 0.05) in leptin-treated cells at 24 h but not at 6 h. The 24 h MMP2 response was greatest (P < 0.05) at 250 ng/mL. Transcript abundance for MMP9 was not altered by leptin treatment. In a separate set of studies, cell proliferation assays were completed. Leptin supplementation did not affect bovine trophoblast cell line proliferation at any dose tested. In conclusion, leptin supplementation did not affect bovine trophoblast cell proliferation or IFNT expression, but leptin increases CSH2 and MMP2 transcript abundance. Both of these factors are involved with peri-implantation and postimplantation placental development and function, and this implicates leptin as a potential mediator of early placental development and function in cattle.

Plane of nutrition affects growth rate, organ size and skeletal muscle satellite cell activity in newborn calves.

Plane of nutrition effects on body, tissue and cellular growth in the neonatal calf are poorly understood. The hypothesis that a low plane of nutrition (LPN) would limit skeletal muscle size by reducing fibre growth and muscle progenitor cell activity was tested. At birth, calves were randomly assigned to either a LPN (20% CP, 20% fat; GE=1.9 Mcal/days) or a high plane of nutrition (HPN; 27% CP, 10% fat, GE = 3.8 Mcal/days) in a 2 × 3 factorial design to test the impact of diet on neonatal calf growth, organ weight and skeletal muscle morphometry with time. Groups of calves (n = 4 or 5) were euthanised at 2, 4 and 8 week of age and organ and empty carcass weights were recorded. Body composition was measured by DXA. Longissimus muscle (LM) fibre cross-sectional area (CSA), fibre/mm2 and Pax7 were measured by immunohistology. Satellite cells were isolated at each time point and proliferation rates were measured by EdU incorporation. Calves fed a HPN had greater (p < 0.05) BW, ADG and hip height than those fed a LPN for 2, 4 or 8 weeks. HPN calves contained a greater (p < 0.05) percentage of fat tissue than LPN calves. Liver, spleen and thymus weights were less (p < 0.05) in LPN calves than HPN animals. Calves fed HPN had larger (p < 0.05) LM CSA at 8 weeks than LPN fed animals with no differences between the groups in numbers of satellite cells per fibre. Proliferation rates of satellite cells isolated from HPN fed calves were greater (p < 0.05) at 2 weeks than LPN fed animals, which exhibited greater (p < 0.05) proliferation rates at 4 weeks than HPN fed calves. We conclude a LPN diet reduces body growth and organ size and metabolically reprograms satellite cell activity.

Effects of recombinant bovine somatotropin administration at breeding on cow, conceptus, and subsequent offspring performance of beef cattle.

The effects of administration of recombinant bovine ST (bST) on plasma hormone concentrations of cows, conceptus development, and postnatal calf performance were examined. Lactating beef cows ( = 190) were exposed to a fixed-time AI (TAI) protocol from d -10 to 0 (TAI on d 0). Cows were blocked by breed and stratified by days postpartum and then randomly assigned to receive, subcutaneously 1) 2 injections of saline (1 mL of 0.9% saline), 1 on d 0 at TAI and a second injection on d 14 (CTRL; = 53); 2) an injection of 325 mg of bST on d 0 and a saline injection on d 14 (bST0; = 48); 3) a saline injection on d 0 and an injection of 325 mg of bST on d 14 (bST14; = 49); or 4) 2 injections of 325 mg of bST, 1 on d 0 and a second injection on d 14 (bST0+14; = 40). Pregnancy status, crown-to-rump length (CRL) on Day 35, and crown-to-nose length (CNL) on Day 65 were determined via transrectal ultrasonography. Blood samples were collected on d 0, 7, 14, 21, 35, and 65, relative to TAI, to determine plasma concentrations of progesterone (P4), IGF-1, and pregnancy-specific protein B (PSPB) and also on d 18 and 21 for isolation of peripheral blood leukocytes for RNA extraction and measurement of interferon-stimulated genes transcript abundance. Individual calf BW was determined at birth and every 30 d until weaning. A subset of 24 calves was randomly selected for liver biopsies at birth to determine mRNA expression of target genes. Administration of bST to cows increased ( < 0.0001) concentrations of plasma IGF-1 for 14 d after injection compared with CTRL but did not affect fetal CRL and CNL ( = 0.23). Cows receiving bST only on d 0 had a greater ( = 0.05) transcript abundance in myxovirus resistance 2 on d 21 compared with 2bST cows (2.0- and 0.8-fold for bST0 and 2bST, respectively), whereas cows receiving bST14 and CTRL were intermediate (1.2- and 0.9-fold, respectively). Calf BW did not differ ( ≥ 0.100) among treatments on d 0, 30, 60, 90, 120, and 150 relative to birth. Injection of bST only on d 0 tended ( = 0.062) to increase calf liver mRNA expression of at birth compared with the calves born to cows in other treatments. Therefore, during a TAI protocol, the administration of 1 or 2 injections of 325 mg of bST to lactating beef cows enhanced their plasma concentrations of IGF-1 but failed to improve fetal size and plasma concentrations of maternal PSPB and P4 and had no effect on postnatal calf growth performance.

Conceptus development and transcriptome at preimplantation stages in lactating dairy cows of distinct genetic groups and estrous cyclic statuses.

The objectives were to compare development and transcriptome of preimplantation conceptuses 15 d after synchronized ovulation and artificial insemination (AI) according to the genetic background of the cow and estrous cyclicity at the initiation of the synchronization program. On d 39±3 postpartum, Holstein cows that were anovular (HA; n=10), Holstein cows that were estrous cyclic (HC; n=25), and Jersey/Holstein crossbred cows that were estrous cyclic (CC; n=25) were randomly selected in a grazing herd and subjected to the Ovsynch protocol. All cows were inseminated on d 49±3 postpartum, which was considered study d 0. Blood was sampled and analyzed for concentrations of progesterone, estradiol, insulin, and insulin-like growth factor 1 (IGF-1) on study d -10, -3, -1, 7, and 15 relative to AI. On study d 15, uteri were flushed and recovered fluid had IFN-τ concentrations measured and subjected to metabolomic analysis. Morphology of the recovered conceptuses was evaluated, and mRNA was extracted and subjected to transcriptome microarray analysis. Compared with HC, CC presented greater concentrations of progesterone and estradiol in plasma, with corpora lutea and preovulatory follicles of similar size. Conceptuses from CC were larger, tended to secrete greater amounts of IFN-τ, and had greater transcript expression of peroxisome proliferator-activated receptor gamma (PPARγ), an important transcription factor that coordinates lipid metabolism and elongation at preimplantation development. In addition, pregnant CC had greater concentrations of anandamide in the uterine flush, which might be important for elongation of the conceptus and early implantation. Conceptuses from HA were also longer and secreted greater amounts of IFN-τ than conceptuses from HC, likely because of the distinct progesterone profiles before and after AI. Nonetheless, anovular cows had reduced concentrations of IGF-1 in plasma, and their conceptuses presented remarkable transcriptomic differences. Some of the altered transcripts suggest that conceptus cells from anovular cows might be under greater cellular stress and presented markers suggesting increased apoptosis and autophagy, which could lead to increased mortality after d 15 of development. Estrous cyclicity had more impact on transcriptome of bovine conceptus than genetic background, and the developmental changes observed during the preimplantation period might be linked to differences in fertility among groups.

Associations between pregnancy-associated glycoproteins and pregnancy outcomes, milk yield, parity, and clinical diseases in high-producing dairy cows.

Pregnancy-associated glycoproteins (PAG) are produced by the ruminant placenta and secreted into the maternal circulation throughout pregnancy. The extent to which circulating PAG concentrations predict pregnancy outcomes was examined herein. Also, associations between circulating PAG concentrations and various production parameters and clinical diseases were evaluated. Lactating primiparous and multiparous Holstein cows (n=345) were bred via timed artificial insemination using a standard Ovsynch protocol. Pregnancy was diagnosed by transrectal ultrasonography at d 32, 46, and 74 of gestation. Blood was harvested at d 32 to determine plasma concentrations of PAG and progesterone. Cows pregnant at d 32 that subsequently lost their pregnancy at d 46 and 74 had reduced PAG concentrations. Both artificial insemination service number and parity were associated with plasma PAG concentrations. Concentration of PAG in plasma was greater for cows pregnant from their second or later breeding than those pregnant from the first breeding postpartum, and was increased for primiparous compared with multiparous. In addition, cows with greater milk yield had increased plasma PAG concentrations. No association was detected between body condition score and plasma PAG concentrations. Cows that experienced clinical metritis, metabolic problems, or left displacement abomasum in the early postpartum period preceding breeding had greater plasma PAG concentrations than cows not experiencing these clinical diseases. Also, cows with multiple clinical diseases had increased odds of pregnancy loss when compared with cows not experiencing clinical diseases. Odds ratio testing detected a tendency in the relationship between reduced milk yield and increased pregnancy loss. Collectively, these associations illustrate one feature of the early developing placenta that may predict pregnancy outcomes in dairy cattle. It is unclear if plasma PAG are actively involved with mediating pregnancy outcomes, but modifications in circulating PAG concentrations due to pregnancy loss, milk yield, parity, and clinical disease implicate placental PAG production or PAG release as being responsive to various physiological stimuli.

Validation of an interferon stimulatory response element reporter gene assay for quantifying type I interferons.

The goal of this work was to develop a virus-free, cell-based interferon (IFN) bioassay and determine the utility of this assay on biological samples that contained IFN-τ, the trophoblast-secreted maternal recognition of pregnancy factor in ruminants. Madin-Darby bovine kidney cells were transduced with lentiviral particles that contained a firefly luciferase reporter construct driven by an IFN stimulatory response element (ISRE). Stably transduced cells were selected with the use of puromycin resistance. A linear, dose-responsive response was detected with human IFN-α and ovine IFN-τ. Interferon activity was detected in conditioned media from bovine trophoblast cells and uterine flushes collected from sheep and cattle. Activity also was detected in media collected after individual or small group culture of in vitro-produced bovine blastocysts at day 8 to 10 after fertilization. In summary, this IFN stimulatory response element-reporter assay may be used as an alternative to virus-dependent, cytopathic assays. It contains a similar sensitivity to IFNs and can be completed in a shorter time than cytopathic assays and does not require heightened biosafety conditions after cell transduction.

Paradoxical effect of supplementary progesterone between Day 3 and Day 7 on corpus luteum function and conceptus development in cattle.

The aim of the present study was to investigate the effect of short-term progesterone (P4) supplementation during the early metoestrous period on circulating P4 concentrations and conceptus development in cattle. The oestrous cycles of cross-bred beef heifers were synchronised using a 7-day P4-releasing intravaginal device (PRID® Delta; 1.55 g P4) treatment with administration of a prostaglandin F(2α) analogue (Enzaprost; CEVA Sante Animale) the day before PRID® Delta removal. Only those heifers recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to one of five groups: (1) control: no treatment; (2) placebo: insertion of a blank device (no P4) from Day 3 to Day 7; (3) insertion of a PRID® Delta from Day 3 to Day 7; (4) insertion of a PRID® Delta from Day 3 to Day 5; or (5) insertion of a PRID® Delta from Day 5 to Day 7. In vitro-produced blastocysts were transferred to each heifer in Groups 2-5 on Day 7 (n=10 blastocysts per heifer) and conceptuses were recovered when heifers were killed on Day 14. Based on the outcome of Experiment 1, in Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to one of three treatment groups: (1) placebo; (2) PRID from Day 3 to Day 5; or (3) PRID from Day 3 to Day 7. All heifers were killed on Day 16 and recovered conceptuses were incubated in synthetic oviducal fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-τ (IFNT). In both experiments, daily blood samples were taken to determined serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Insertion of a PRID resulted in an increase (P<0.05) in serum P4 that declined following removal. In Experiment 1, P4 supplementation from Day 3 to Day 5 (17.0±1.4 mm) or Day 3 to Day 7 (11.3±2.3 mm) increased conceptus length compared with placebo (2.1±1.8 mm). Serum P4 was significantly lower from Day 9 to Day 14 (P<0.05) and the weight of the Day 14 corpus luteum (CL) was lower in the PRID Day 3-7 group than the placebo or control groups. In Experiment 2, supplementation from Day 3 to Day 5 (94.0±18.8 mm) or Day 3 to Day 7 (143.6±20.6 mm) increased conceptus length on Day 16 compared with placebo (50.3±17.4 mm). Serum P4 was significantly lower in the two supplemented groups following PRID removal compared with placebo (P<0.05) and was associated with a lower CL weight in the Day 3-7 group. Conceptus length was strongly correlated with the IFNT concentration in the uterine flush (r=0.58; P=0.011) and spent culture medium (r=0.68; P<0.002). The findings of the present study highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrous period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan.

Subspecies differences in early fetal development and plasma pregnancy-associated glycoprotein concentrations in cattle.

Inclusion of Bos indicus genetics improves production traits of cattle maintained in hot climates. Limited information exists detailing pregnancy-specific events as influenced by variable amounts of Bos indicus genetics. Three experiments were completed to examine the effect of Bos taurus and Bos indicus genotypes on fetal size and plasma pregnancy-associated glycoprotein (PAG) concentrations. In all experiments, cows were bred by AI after synchronization of ovulation. Fetal measurements were completed by transrectal ultrasonography and plasma PAG concentrations were quantified from plasma harvested the day of each fetal measurement. In Exp. 1, fetal size and plasma PAG concentrations were measured at d 53 of pregnancy in cows composed of various fractions of Angus and Brahman (n = 9 to 21 cows/group). Fetus size was greater in cows containing >80% Angus genetics compared with cows containing <80% Angus influence (3.40 ± 0.28 vs. 2.86 ± 0.28 cm crown-rump length; P < 0.01). Plasma PAG concentrations were reduced (P < 0.01) in cows containing >80% Angus genetics when compared with their contemporaries (6.0 ± 1.5 ng/mL vs. 9.4 ± 1.5 ng/mL). In Exp. 2, fetal measurements and plasma PAG concentrations were determined at d 35 and 62 of pregnancy in Angus and Brangus cows. Breed did not affect fetus size at d 35, but Angus cows contained larger fetuses than Brangus cows at d 62 [3.0 ± 0.03 vs. 2.8 ± 0.03 cm crown-nose length (CNL; P > 0.01)]. Plasma PAG concentrations were not different between breed at d 35 and 62 (P > 0.1). In Exp. 3, fetal measurements and plasma samples were collected at d 33/34, 40/41, 47/48, and 54/55 post-AI in Angus and Brangus cows. Fetus size was not different (P > 0.05) between genotypes on d 33/34, 40/41, and 47/48. Angus fetuses were larger than Brangus fetuses at d 54/55 (2.1 ± 0.03 vs. 1.9 ± 0.03 cm CNL; P = 0.001). Plasma PAG concentrations were less in Angus than Brangus cows at each time point (average 4.9 ± 0.9 vs. 8.2 ± 0.9 ng/mL; P = 0.005). In conclusion, these studies determined that the Bos taurus × Bos indicus genotype impacts fetal size and rate of fetal development by 7 wk of gestation. Plasma PAG concentrations were increased in cattle with Bos indicus genetics in 2 of 3 studies, suggesting that genotype is one of several determinants of PAG production and secretion in cattle.

Environmental regulation of pregnancy-specific protein B concentrations during late pregnancy in dairy cattle.

Environmental factors, such as photoperiod and heat stress, can be manipulated during the dry period to influence health, productivity, and reproductive performance of dairy cows in their subsequent lactation. The impacts of photoperiod and heat stress on subsequent lactation are related to alterations in prolactin (PRL) signaling and may affect the expression of pregnancy-specific protein B (PSPB). Additionally, exposure of cows to heat stress during the dry period decreases gestation length; however, the mechanism involved in this process is unknown. The objective of these experiments was to evaluate the influence of environmental factors (i.e., heat stress and photoperiod) during late gestation (i.e., dry period) on PSPB concentrations in plasma of dairy cows. In Exp. 1, cows were dried off in the summer months approximately 46 d before expected calving and assigned randomly to heat stress (HT; n=30) or cooling (CL; n=30) treatment. Cooling cows were housed with sprinklers, fans, and shade, whereas HT cows were provided only shade. In Exp. 2, cows were dried off at approximately 60 d before expected calving in summer/fall months and randomly assigned to 3 treatments: long day photoperiod (LDPP: 16L:8D; n=15), short day photoperiod (SDPP: 8L:16D; n=14) and SDPP+PRL implant (12 mg/d of PRL at 28 d or 16 mg/d of PRL at 39 d; n=11). In both experiments, plasma samples were collected at dry off and at -32, -18, -7, -3 and 0 d relative to calving. In Exp. 1, greater concentrations of PSPB were detected in plasma of CL versus HT cows (388.3±24.7 vs. 287.4±23.8 ng/mL; P<0.01). Concentrations of PSPB did not differ between -46 to -18 d before calving (66.0 ng/mL). However, PSPB concentrations were greater (P<0.01) for CL cows at d -7 (534.7>357.2 ng/mL), -3 (807.2>572.2 ng/mL) and 0 (800.8>563.5 ng/mL) relative to calving. Additionally, HT cows in Exp. 1 had increased PRL plasma concentrations compared with CL cows (21.01±1.6 vs. 13.78±1.6 ng/mL). In Exp. 2, no differences were detected in plasma concentrations of PSPB (ng/mL) among LDPP, SDPP, or SDPP+PRL groups on d -60 (41.5), -32 (51.7), -18 (58.5), -7 (532.9), -3 (838.2), and 0 (729.4) relative to parturition. Photoperiodic PRL concentrations were 10.81, 7.84, and 4.22 ng/mL for LDPP, SDPP+ PRL, and SDPP, respectively. Results indicate that HT alters PSPB concentrations in late pregnancy, suggesting that placental activity is altered in cows exposed to excessive elevated temperatures around the time of calving. However, the mechanism involved likely is not associated with changes in PRL secretion.

Effects of lactation and pregnancy on gene expression of endometrium of Holstein cows at day 17 of the estrous cycle or pregnancy.

Objectives were to determine effects of lactation and pregnancy on endometrial gene expression on d 17 of the estrous cycle and pregnancy. Heifers (n=33) were assigned randomly after parturition to lactating (L, n=17) or nonlactating (NL, n=16) groups. Cows were subjected to an ovulation synchronization program for a timed artificial insemination (TAI); 10 cows in L and 12 in NL were inseminated. Slaughter occurred 17 d after the day equivalent to TAI, and intercaruncular endometrial tissues were collected. Gene expression was determined by DNA microarray analysis for pregnant (L, n=8; NL, n=6) and noninseminated cyclic (L, n=7; NL, n=4) cows. Differentially expressed genes were selected with a P-value <0.01 and absolute expression >40. In addition, a fold effect >1.5 was used as a criterion for genes affected by pregnancy. In total, 210 genes were differentially regulated by lactation (136 downregulated and 74 upregulated), and 702 genes were differentially regulated by pregnancy (407 downregulated and 295 upregulated). The interaction effect of pregnancy and lactation affected 61 genes. Genes up- and downregulated in pregnant cows were associated with several gene ontology terms, such as defense response and interferon regulatory factor, cell adhesion, and extracellular matrix. The gene ontology analyses of up- and downregulated genes of lactating cows revealed terms related to immunoglobulin-like fold, immune response, COMM domain, and non-membrane-bounded organelle. Several genes upregulated by lactation, such as IGHG1, IGLL1, IGK, and TRD, were related to immune function, particularly for B cells and γδ T cells. Developmental genes related to limb and neural development and glucose homeostasis (e.g., DKK1, RELN, PDK4) were downregulated by lactation, whereas an interaction was also detected for RELN. The stated genes associated with immune function and developmental genes expressed in the endometrium affected by lactational state are possible candidate genes for interventions to improve fertility of lactating dairy cows.

Effects of lactation and pregnancy on metabolic and hormonal responses and expression of selected conceptus and endometrial genes of Holstein dairy cattle.

Objectives were to characterize postpartum metabolic and hormonal differences between nonlactating and lactating dairy cows, evaluate lactation and pregnancy effects on endometrium and conceptus expression of selected genes, and characterize associations between conceptus and endometrial expression of genes in early pregnancy (d 17). Pregnant heifers were assigned randomly after calving to a lactating group (L, n=17) and a nonlactating group (NL, n=16). The L cows were fed a total mixed ration [1.65 Mcal of net energy for lactation (NE(L))/kg, 16.5% crude protein (CP)] ad libitum, and the NL cows were fed a maintenance ration (1.45 Mcal of NE(L)/kg, 12.2% CP) once per day. All cows were presynchronized and enrolled in a timed artificial insemination (TAI) protocol; 10 cows in the L and 12 in the NL received TAI. On d 17 after GnRH and TAI, cows were slaughtered and endometrial and conceptus tissues collected. The Affymetrix Bovine Genome DNA Microarray (Affymetrix Inc., Santa Clara, CA) was used to assess conceptus and endometrial gene expression. The L cows had higher body temperature than the NL cows (38.4 vs. 38.2°C), and the NL cows cycled earlier than the L cows (26.3 vs. 34.7 d in milk). Cows in the L group had greater plasma concentrations of ß-hydroxybutyrate (4.90 vs. 2.97 mg/dL) and blood urea N (11.6 vs. 6.5mg/dL) and lower concentrations of glucose (74.0 vs. 79.9 mg/dL) compared with NL cows. Insulin-like growth factor-1 was lower for L compared with NL (140.5 vs. 198.2 ng/mL) and was greater for cows subsequently classified pregnant compared with cyclic (191.0 vs. 147.6 ng/mL). The concentration of progesterone from GnRH or TAI (d 0) until d 17 was lower for L cows than for NL cows. Gene expression analyses indicated that all conceptuses (n=13) expressed pregnancy-associated glycoprotein (PAG) genes PAG2, PAG8, PAG11, and PAG12. The same PAG family genes were observed in the endometrium of some pregnant cows. Simple and standard partial correlation analyses detected associations of conceptus PAG11 with prostaglandin regulatory genes. In conclusion, lactation altered metabolic status, delayed initiation of cyclicity, and decreased concentrations of progesterone in pregnant cows. Early expression of PAG genes in the conceptus may contribute to successful development of early pregnancy and possibly alter mechanisms related with embryo survival such as prostaglandin synthesis.

Effects of human chorionic gonadotrophin administration on day 5 after oestrus on corpus luteum characteristics, circulating progesterone and conceptus elongation in cattle.

The aim of the present study was to test the hypothesis that elevated concentrations of progesterone (P4) resulting from the induction of an accessory corpus luteum (CL) by human chorionic gonadotrophin (hCG) administration on day 5 after oestrus would lead to advanced conceptus elongation on day 14 following embryo transfer on day 7. The oestrous cycles of cross-bred beef heifers were synchronised and animals were randomly assigned to receive either of two treatments: (1) intramuscular injection of 3000 IU hCG on day 5 after oestrus (n=14); or (2) intramuscular injection of saline on day 5 after oestrus (n=13). Ovaries were scanned daily by transrectal ultrasonography to assess CL development. Serum concentrations of P4 were determined from daily blood samples collected from the jugular vein. In vitro-produced bovine blastocysts were transferred to synchronised recipients on day 7 after oestrus (n=15 blastocysts per recipient). Heifers were killed on day 14 after oestrus and the uterus was flushed to recover the embryos. Injection of hCG on day 5 induced ovulation of the dominant follicle in all treated heifers and increased the total area of luteal tissue on the ovary, which was associated with a significant increase (P<0.001) in serum concentrations of P4 from day 7 to day 14. Positive associations were detected between circulating P4 with CL area (within-day correlations ranging from r=0.45 to r=0.67) and total area of luteal tissue (within-day correlations ranging from r=0.65 to r=0.86) Administration of hCG did not affect the proportion of day 14 conceptuses recovered. However, compared with the control group, hCG-treated heifers had increased conceptus length (3.91±1.23 vs. 5.57±1.02 mm, respectively; P=0.06), width (1.00±0.06 vs. 1.45±0.05 mm, respectively; P=0.002) and area (5.71±0.97 vs. 8.31±0.83, respectively; P=0.02). Although numerically greater, mean interferon-τ (IFNT) production in vitro did not differ significantly (P=0.54) between embryos recovered from hCG-treated and control heifers. In contrast, there was a strong positive correlation between individual embryo length (r=0.76; P<0.001) and individual embryo area (r=0.72; P<0.001) and IFNT production. In conclusion, administration of hCG on day 5 after oestrus resulted in the formation of an accessory CL and hypertrophy of the original CL, the result of which was an increase in P4 concentrations from day 7 onwards. These elevated P4 concentrations were associated with an increased conceptus area. Furthermore, conceptus size was highly correlated with IFNT secretion in vitro.

Disruption of fibroblast growth factor receptor signaling in bovine cumulus-oocyte complexes during in vitro maturation reduces subsequent embryonic development.

Several fibroblast growth factors (FGF) mediate folliculogenesis and oogenesis in cattle but it is unclear whether FGFs are required during the final stages of oocyte maturation. The objectives of this work were to determine whether blocking FGF receptor (FGFR) activity during in vitro maturation (IVM) affects oocyte fertilization and embryo development; examine changes in FGFR transcript profiles in cumulus cells and oocytes during IVM; and evaluate whether gonadotropins modulate FGFR transcript abundance during IVM. In the first set of studies, bovine cumulus-oocyte complexes (COCs) were matured in the presence of one of two FGFR kinase inhibitors (SU5402 or PD173074). After maturation, COCs were washed and cultured without inhibitors. Inhibitors did not affect cleavage rates but the percentage of ≥ 8-cell embryos at d 3 and blastocysts at d 7 and d 8 postfertilization were decreased when COCs were matured with either inhibitor. Profiles of FGFR mRNA variants were examined in cumulus cells and oocytes separated either immediately before (0 h) or at 6 or 21 h after beginning IVM. In cumulus cells, increases in R1b, R2b, and R2c abundance were detected when cultured in the absence of follicle-stimulating hormone (FSH). Supplementing FSH (1 or 25 µM) increased the abundance of R1b, R1c, R2b, and R2c. In oocytes, no time- or FSH-dependent changes in FGFR transcript abundance were detected. These observations implicate FGFs as crucial components of bovine oocyte competency and indicate that FSH augments FGFR mRNA abundance in cumulus cells during the final stages of oocyte maturation.

Conceptus-mediated endometrial vascular changes during early pregnancy in mares: an anatomic, histomorphometric, and vascular endothelial growth factor receptor system immunolocalization and gene expression study.

This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation.