Review: Reproductive consequences of whole-body adaptations of dairy cattle to heat stress.

Heat stress has far-reaching ramifications for agricultural production and the severity of its impact has increased alongside the growing threats of global warming. Climate change is exacerbating the already-severe consequences of seasonal heat stress and is predicted to cause additional losses in reproductive performance, milk production and overall productivity. Estimated and predicted losses are staggering, and without advancement in production practices during heat stress, these projected losses will threaten the human food supply. This is particularly concerning as the worldwide population and, thus, demand for animal products grows. As such, there is an urgent need for the development of technologies and management strategies capable of improving animal production capacity and efficiency during periods of heat stress. Reproduction is a major component of animal productivity, and subfertility during thermal stress is ultimately the result of both reproductive and whole-body physiological responses to heat stress. Improving reproductive performance during seasonal heat stress requires a thorough understanding of its effects on the reproductive system as well as other physiological systems involved in the whole-body response to elevated ambient temperature. To that end, this review will explore the reproductive repercussions of whole-body consequences of heat stress, including elevated body temperature, altered metabolism and circulating lipopolysaccharide. A comprehensive understanding of the physiological responses to heat stress is a prerequisite for improving fertility, and thus, the overall productivity of dairy cattle experiencing heat stress.

Human continuous glucose monitors for measurement of glucose in dairy cows.

If validated for use in dairy cattle, interstitial continuous glucose monitors (CGMs) could be easily implemented, informative tools for research, clinical, and perhaps even on-farm applications. To evaluate their efficacy, 2 experiments were conducted, during which lactating Holstein cows were fit with indwelling jugular catheters, as well as FreeStyle Libre (FSL; Abbott) and Dexcom G6 (DexCom Inc.) CGMs secured either behind their polls, lateral to their ears, or beneath their pin bones on their upper rear legs. During the first experiment, blood (measured with a handheld glucometer) and interstitial glucose measurements were collected from 13 cows every 4 h for 96 h. In the second experiment, the same measurements were collected from 8 cows every 15 min for 6 h. At the mid-point of the sampling period (3 h), cows received a bolus dose of dextrose to facilitate comparisons across a broad range of glucose concentrations. Results from both experiments determined that functional longevity of the sensors was greatest for those sensors secured near the ear. Likewise, interstitial measurements from the ear sensors were most closely correlated with blood glucose concentrations (r = 0.82 and r = 0.71 for FSL ear and Dexcom G6 ear, respectively). Unfortunately, accuracy calculated as absolute relative error was low, at 60.7% or less. As a result of the low accuracy, even though both ear sensors detected an increase in glucose concentrations following the bolus dose, neither produced results exactly matching blood glucose measurements. The results of this work indicate that the FSL and Dexcom G6 CGMs are not currently capable of replacing blood-based glucose measurements.

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 periconceptional heat stress on primiparous and multiparous daughters of Holstein dairy cows.

To meet growing worldwide demands for animal products, animal production will need to increase in capacity and efficiency. Every opportunity to improve animal protein yield should be considered and explored. Developmental programming is one such opportunity that has not yet been thoroughly investigated in farm animal production. While developmental programming can be advantageous for the survival of the offspring, it is often described in conjunction with negative consequences. The known and potential causes and mechanisms are numerous, often stemming from some sort of stress experienced during the prenatal or early postnatal period. One stressor that is particularly concerning for farm animal production is heat stress. Heat stress is known to elicit adaptations associated with developmental programming in several species, but has not been investigated in dairy cattle until recently. Multiple studies have shown that heat stress experienced during the periconceptional period is generally associated with reduced milk production of resulting offspring. This could be the result of adaptations within the pre-ovulatory oocyte or early developing embryo. Interestingly, in a few select comparisons, periconceptional heat stress was associated with greater milk production. This was only observed when dairy cattle calved in the spring, and would therefore be reaching peak milk production in late spring or early summer (in heat stress). This is consistent with the match/mismatch theory associated with developmental programming, where matched prenatal/postnatal environments confer advantageous adaptations and mismatched prenatal/postnatal environments are generally detrimental to the offspring. While these studies are important additions to our growing knowledge of heat stress impacts on dairy cow production, the broader implication of developmental programming requires further investigation.

Invited review: Management strategies capable of improving the reproductive performance of heat-stressed dairy cattle.

Impaired fertility during periods of heat stress is the culmination of numerous physiological responses to heat stress, ranging from reduced estrus expression and altered follicular function to early embryonic death. Furthermore, heat-stressed dairy cattle exhibit a unique metabolic status that likely contributes to the observed reduction in fertility. An understanding of this unique physiological response can be used as a basis for improving cow management strategies, thereby reducing the negative effects of heat stress on reproduction. Potential opportunities for improving the management of dairy cattle during heat stress vary greatly and include feed additives, targeted cooling, genetic selection, embryo transfer and, potentially, crossbreeding. Previous studies indicate that dietary interventions such as melatonin and chromium supplementation could alleviate some of the detrimental effects of heat stress on fertility, and that factors involved in the methionine cycle would likely do the same. These supplements, particularly chromium, may improve reproductive performance during heat stress by alleviating insulin-mediated damage to the follicle and its enclosed cumulus-oocyte complex. Beyond feed additives, some of the simplest, yet most effective strategies involve altering the timing of feeding and cooling to take advantage of comparatively low nighttime temperatures. Likewise, expansion of cooling systems to include breeding-age heifers and dry cows has significant benefits for dams and their offspring. More complicated but promising strategies involve the calculation of breeding values for thermotolerance, the identification of genomic markers for heat tolerance, and the development of bedding-based conductive cooling systems. Unfortunately, no single approach can completely rescue the fertility of lactating dairy cows during heat stress. That said, region-appropriate combinations of strategies can improve reproductive measures to reasonable levels.

Metabolic and reproductive characteristics of replacement beef heifers subjected to an early-weaning regimen involving high-concentrate feeding.

In an effort to better understand the consequences of early weaning (EW) for replacement beef heifers, a two-phase experiment was conducted investigating the impact on metabolic function and documenting reproductive characteristics. In phase 1, Angus×Simmental heifers (n=35) were stratified by BW and sire, and randomly assigned to either a normal weaning (NW, n=18) or EW (n=17) treatment. EW heifers were weaned at 107±3 days of age and provided access to a concentrate-based ration ad libitum with limit-fed mixed grass hay. NW heifers remained with their dams until 232±3 days of age, at which point heifers from both treatments were comingled and grazed on mixed summer pasture. Following NW, weekly blood samples were collected from all heifers for progesterone analyses used to determine the onset of puberty. Pelvic and ovarian size was measured before breeding. All heifers were subjected to an estrous synchronization protocol with timed artificial insemination (AI) at 437±4 days of age. During phase 2 of the experiment, a subset of pregnant heifers (n=16) were divided into two replicates and subjected to a glucose tolerance test, epinephrine challenge and progesterone clearance analysis. Neither age nor BW at puberty differed between EW and NW heifers. Likewise, no differences in pelvic area or ovarian size were observed. Thus, it appears that the reproductive maturity of EW and NW heifers was similar. Heifers studied during phase 2 of the experiment were restricted to those that had become pregnant to their first AI. Within this cohort, EW heifers tended to have lower overall circulating progesterone concentrations than those that were NW (P=0.14). Aspects of glucose and insulin dynamics were also altered, as EW heifers tended to have lower baseline glucose concentrations (P=0.10) despite similar baseline insulin concentrations. Compared with NW heifers, EW heifers had lower insulin area under the curve (P<0.05), which was partly the result of a tendency for lower peak insulin concentrations (P=0.11). Results of the glucose tolerance test indicate that a lesser insulin response was necessary to properly clear the glucose in the EW heifers, suggesting enhanced insulin sensitivity. Collectively, these results indicate that EW is not detrimental for the growth or reproductive development of replacement beef heifers, although some differences in glucose and insulin dynamics persist into adulthood.

Immunological castration temporarily reduces testis size and function without long-term effects on libido and sperm quality in boars.

The objective was to determine the effects of immunization against gonadotropin-releasing hormone on reproductive characteristics in boars. A total of 72 boars were used in a randomized design with three treatments: single immunization (SI) (10 weeks of age) or double immunization (DI) (10 and 15 weeks of age) with Improvest® and intact controls (no Improvest®; CNT) (n=24/group). At 10, 15, 20, 25 and 40 weeks of age, blood was collected and serum harvested to evaluate testosterone concentrations. Testosterone concentrations were less for DI boars compared with CNT boars and SI boars at 20 and 25 weeks (P<0.001), but not at 40 weeks of age. At week 25, 18 pigs (n=6/group) were sacrificed and testes were removed, weighed and measured, and seminiferous tubules were examined and scored using histological slides of testes parenchyma. A sample of neck fat was assessed for boar taint aroma. All testicular measurements and weights and seminiferous tubule scores were less for DI boars compared with SI and CNT boars (P<0.001). More (P<0.05) SI and CNT boars had detectable boar taint aroma than DI boars. Libido was assessed at 32, 36, 47, 60 and 63 weeks of age and semen collected at 60 weeks of age was analyzed for indicators of quality. There were no effects of treatment (P=0.41) or treatment by week (P=0.71) on libido. Semen volume, gel weight and total number of sperm cells, determined in a subset of boars (n=3/treatment), were not different among treatments. Sperm concentration was greater for DI than SI (P=0.01), and tended to be greater for DI compared with CNT (P=0.10). Sperm motility tended to be greater for DI boars compared with CNT boars (P=0.066). In conclusion, our results show that there are no long-term effects of immunocastration on reproductive characteristics in boars.

Prepubertal tamoxifen treatment affects development of heifer reproductive tissues and related signaling pathways.

Prepubertal exposure of the developing ovaries and reproductive tract (RT) to estrogen or xenoestrogens can have acute and long-term consequences that compromise the reproductive performance of cattle. This research examined effects of the selective estrogen receptor modulator tamoxifen (TAM) on gene and protein abundance in prepubertal ovaries and RT, with a particular focus on signaling pathways that affect morphology. Tamoxifen was administered to Holstein heifer calves (n=8) daily (0.3mg/kg subcutaneously) from 28 to 120 d of age, when tissues were collected. Control calves (n=7) received an equal volume of excipient. Weight, gross measurements, and samples of reproductive tissues were collected, and protein and mRNA were extracted from snap-frozen samples of vagina, cervix, uterus, oviduct, ovary, and liver. Neither estradiol nor insulin-like growth factor I (IGFI) concentrations in the serum were affected by TAM treatment. Tamoxifen treatment reduced ovarian weight independently from effects on antral follicle populations, as there was no difference in visible antral follicle numbers on the day of collection. Estrogen receptor α (ESR1) and ß (ESR2) mRNA, ESR1 protein, IGFI, progesterone receptor, total growth hormone receptor, WNT4, WNT5A, and WNT7A mRNA, in addition to mitogen-activated protein kinase (MAPK) and phosphorylated MAPK proteins were affected differently depending on the tissue examined. However, neither IGFI receptor mRNA nor protein abundance were affected by TAM treatment. Results indicate that reproductive development in prepubertal Holstein heifer calves is TAM-sensitive, and that bovine RT and ovarian development are supported, in part, by estrogen receptor-dependent mechanisms during the period studied here. Potential long-term consequences of such developmental disruption remain to be defined.

Skeletal muscle and hepatic insulin signaling is maintained in heat-stressed lactating Holstein cows.

Multiparous cows (n=12; parity=2; 136±8 d in milk, 560±32kg of body weight) housed in climate-controlled chambers were fed a total mixed ration (TMR) consisting primarily of alfalfa hay and steam-flaked corn. During the first experimental period (P1), all 12 cows were housed in thermoneutral conditions (18°C, 20% humidity) with ad libitum intake for 9 d. During the second experimental period (P2), half of the cows were fed for ad libitum intake and subjected to heat-stress conditions [WFHS, n=6; cyclical temperature 31.1 to 38.9°C, 20% humidity: minimum temperature humidity index (THI)=73, maximum THI=80.5], and half of the cows were pair-fed to match the intake of WFHS cows in thermal neutral conditions (TNPF, n=6) for 9 d. Rectal temperature and respiration rate were measured thrice daily at 0430, 1200, and 1630 h. To evaluate muscle and liver insulin responsiveness, biopsies were obtained immediately before and after an insulin tolerance test on the last day of each period. Insulin receptor (IR), insulin receptor substrate 1 (IRS-1), AKT/protein kinase B (AKT), and phosphorylated AKT (p-AKT) were measured by Western blot analyses for both tissues. During P2, WFHS increased rectal temperature and respiration rate by 1.48°C and 2.4-fold, respectively. Heat stress reduced dry matter intake by 8kg/d and, by design, TNPF cows had similar intake reductions. Milk yield was decreased similarly (30%) in WFHS and TNPF cows, and both groups entered into a similar (-4.5 Mcal/d) calculated negative energy balance during P2. Insulin infusion caused a less rapid glucose disposal in P2 compared with P1, but glucose clearance did not differ between environments in P2. In liver, insulin increased p-AKT protein content in each period. Phosphorylation ratio of AKT increased 120% in each period after insulin infusion. In skeletal muscle, protein abundance of the IR, IRS, and AKT remained stable between periods and environment. Insulin increased skeletal muscle p-AKT in each period, but the phosphorylation ratio (abundance of phosphorylated protein:abundance of total protein) of AKT was decreased in P2 for TNPF animals, but not during WFHS. These results indicate that mild systemic insulin resistance during HS may be related to reduced nutrient intake but skeletal muscle and liver insulin signaling remains unchanged.

Tamoxifen impairs prepubertal mammary development and alters expression of estrogen receptor α (ESR1) and progesterone receptors (PGR).

Research has shown that prepubertal heifers experience allometric mammary growth that is influenced by the ovaries. Our purpose was to determine the role of estrogen in prepubertal mammary gland development. Sixteen Holstein calves were randomly assigned to 1 of 2 treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were administered the antiestrogen tamoxifen (0.3 mg kg(1) d(1)) or placebo from 28 to 120 d of age. At 120 d, calves were euthanized and udders removed. Weight and DNA content of trimmed parenchymal tissue were halved (P ≤ 0.0001) in TAM compared with CON calves. Parenchymal samples from 3 zones of the left rear mammary gland (lower, middle, and outer regions) were processed for immunohistochemical staining for estrogen receptor α (ESR1) and progesterone receptor (PGR), Ki67-positive cells, and 5-bromo-2'-deoxyuridine label retaining cells (LRCs). Overall, neither the percentage nor location within the epithelial tissue layer of either ESR1- or PGR-positive cells was impacted by TAM treatment. However, image analysis indicated a 6.2-fold lower (P = 0.0001) level of ESR1 protein expression in TAM calves. Similarly, messenger RNA expression of ESR1 was also reduced (P = 0.0001) in TAM heifers. In contrast, expression of PGR protein was greater by 43% (P = 0.03) in TAM calves, but messenger RNA expression did not differ between treatments. Overall, TAM calves had a higher (P ≤ 0.03) percentage and density (cells per tissue area) of Ki67-positive cells. Irrespective of treatment, there were also more Ki67-labeled cells in the outer zones of the mammary gland (P ≤ 0.001). We were able to effectively use multispectral imaging to identify positive cells and quantify the expression of ESR1 and PGR protein. We also identified and counted the proportion of label retaining cells (LCR) (putative epithelial stem cells). We noted an overall 2.9-fold greater number of LRCs in TAM heifers and more LRCs in the outer sampling zones. This suggests that a cohort of LCR cells in TAM remained inactivated in comparison with CON heifers, which exhibited markedly increased growth of the mammary parenchymal tissue over the treatment period. These results suggest that the impacts of ovariectomy are partially explained by loss of ESR1 expression and/or estrogen receptor signaling in the prepubertal bovine mammary gland. The significance of mammary expression of PGR in control of prepubertal bovine mammary development remains unresolved.

Short communication: Hepatic progesterone-metabolizing enzymes cytochrome P450 2C and 3A in lactating cows during thermoneutral and heat stress conditions.

Two experiments were performed to determine the effects of heat stress (HS) and insulin on hepatic mRNA abundance of enzymes responsible for metabolizing progesterone [cytochrome P450 2C and 3A (CYP2C and CYP3A)]. To distinguish the direct effects of HS from decreased dry matter intake, cohorts were pair fed (PF) in thermoneutral conditions to match the intake of the HS cows during both experiments. In the first experiment, multiparous late-lactation Holstein cows (n=12, 305±33 d in milk) housed in climate-controlled chambers were subjected to 2 experimental periods: (1) thermoneutral (TN) conditions (18°C, 20% humidity) with ad libitum intake (TN and well fed) for 9 d; and (2) either HS conditions (cyclical temperature 31-40°C, 20% humidity) fed for ad libitum intake (n=6), or TN conditions and PF to match the HS animal (n=6) for 9 d. To evaluate hepatic gene expression during experiment 1, biopsies were obtained at the end of each period. In the second experiment, multiparous mid-lactation Holstein cows (n=12, 136±8 DIM) were housed and fed in conditions similar to those described for the first experiment. Liver biopsies were obtained immediately before and after an insulin tolerance test administered on d 6 of each period. No effects of exogenous insulin were observed on any of the tested variables, nor were there interactions between environment (TN/HS or well fed/PF) and insulin administration. Heat stress decreased hepatic CYP2C expression during both experiments. The relative abundance of CYP3A was not affected by environmental conditions in the late-lactation cows (first experiment), but was reduced by HS in the mid-lactation cows (second experiment). Interestingly, during experiment 2, hepatic CYP3A expression also decreased during PF. These results suggest that HS reduces the capacity of the liver to metabolize progesterone through distinct effects on CYP2C and CYP3A, and that the effects appear to vary based upon stage of lactation. Ultimately, HS may affect reproductive outcomes by reducing the abundance of the enzymes responsible for the breakdown of progesterone. This reduction could serve as a beneficial adaptation for rescuing early embryos or may be detrimental, as it affects feedback mechanisms necessary for proper cyclicity.

Reproductive performance of lactating dairy cattle after intrauterine administration of a prostaglandin F2α receptor antagonist 4 days after insemination.

Previous research has determined that PGF2α detrimentally affects pregnancy via direct effects on early embryonic development. Because early embryonic loss is relatively prevalent in lactating dairy cows, we hypothesized that pregnancy retention (and resulting conception rates) would be improved by administering a PGF2α receptor antagonist (AL-8810) shortly after insemination. Multiparous, lactating Holstein dairy cows were randomly assigned to receive one of four intrauterine treatments: (1) control group-untreated cohort (CON; n = 93); (2) control group-vehicle infusion (CON-V; n = 90); (3) 2000 nM AL-8810 infusion (AL-2000; n = 96); or (4) 10,000 nM AL-8810 infusion (AL-10,000; n = 93). Treatments were administered transcervically 4 days after insemination in the horn ipsilateral to the CL. There was no effect of treatment on conception rate (36.6%, 38.9%, 25.0%, and 35.5% for CON, CON-V, AL-2000, and AL-10,000, respectively) or calving rate (24.7%, 24.4%, 16.7%, and 28.0% for CON, CON-V, AL-2000, and AL-10,000, respectively). There was a significant effect of treatment on return to estrus with CON-V (23.6 ± 0.6) and AL-10,000 (23.3 ± 0.6) groups having a longer interval to next estrus over the CON group (21.5 ± 0.6; P < 0.05). Prior treatment did not affect conception to the subsequent insemination. It is important to note that although the addition of AL-8810 into the uterus on Day 4 after insemination did not increase conception rates in the present experiment, it also did not have a negative impact. Furthermore, the treatment procedure itself did not impair the establishment of pregnancy (CON vs. CON-V, AL-2000, and AL-10,000). These results demonstrate that a therapeutic agent can be administered directly into the uterus on Day 4 after insemination without detrimentally affecting conception rates.

Effects of prolonged nutrient restriction on baseline and periprandial plasma ghrelin concentrations of postpubertal Holstein heifers.

Objectives of this study were to measure both daily and periprandial plasma ghrelin concentrations of postpubertal Holstein heifers during prolonged undernutrition. Following an acclimation period, Holstein heifers [n=10; 339.5 ± 8.6 kg of body weight (BW)] were fed ad libitum [well fed (WF); n=5] or restricted to 50% of ad libitum intake [underfed (UF); n=5) for 8 wk. Body condition scores (BCS) were recorded at the beginning and end of the treatment period, and weekly measurements of BW, plasma ghrelin, progesterone, and nonesterified fatty acids (NEFA) concentrations were obtained. Ovarian follicular and luteal structures were measured twice weekly via transrectal ultrasonography. Plasma ghrelin concentrations were also measured during a periprandial window bleed conducted at the end of the experiment. During the window bleed, samples were collected every 15 min between 0500 and 0900 h, with feed offered at 0700 h. Underfed heifers lost BW and BCS, whereas WF heifers gained weight and either increased or maintained BCS. Chronic underfeeding increased circulating ghrelin and NEFA concentrations. By wk 4 of the treatment period, circulating ghrelin concentrations of the UF heifers reached a plateau. Periprandial fluctuations in ghrelin concentrations were apparent as plasma ghrelin concentrations changed over time. Overall differences in periprandial plasma ghrelin concentrations were primarily due to prefeeding effects of plane of nutrition. Plasma ghrelin concentrations and change in BCS were negatively correlated such that heifers that lost the most BCS had the highest concentrations of circulating ghrelin. Two of the 5 UF heifers became anestrus by wk 3 of the treatment period. Despite being of similar age, the heifers that became anestrus had lower BW and plasma ghrelin concentrations than the UF heifers that continued to ovulate. In the current experiment, long-term undernutrition elicited ghrelin responses similar to those reported for shorter durations of nutrient restriction in cattle and other ruminants. These results demonstrate that plane of nutrition is a chronic regulator of plasma ghrelin concentrations, and that these concentrations can be experimentally manipulated in postpubertal heifers for up to 8 wk with no evidence of an adaptive response.

Localization of ghrelin and its receptor in the reproductive tract of Holstein heifers.

The aim of this experiment was to localize the mRNA and protein of ghrelin and its active receptor, growth hormone secretagogue 1A (GHS-R1A), within the reproductive tract of dairy cattle. Ghrelin is an orexigenic hormone that has been identified as a potent regulator of energy homeostasis. Recent evidence suggests that ghrelin may also serve as a metabolic signal to the reproductive tract. Ghrelin and GHS-R1A have been identified in the reproductive tract of several species, including humans, mice, and rats. However, ghrelin and GHS-R1A expression have not been described within bovine reproductive tissues. Therefore, the ampulla, isthmus, uterine body, corpus luteum, and follicles were harvested from 3 Holstein heifers (15.91±0.07 mo of age) immediately following exsanguination. Duodenum and hypothalamus were collected as positive controls for ghrelin and GHS-R1A, respectively. Tissues were fixed in 10% formalin and embedded in paraffin for microscopy. Additional samples were stored at -80°C for detection of mRNA. Ghrelin and GHS-R1A mRNA and protein were observed in all tissue types within the reproductive tract of dairy heifers; however, expression appeared to be cell specific. Furthermore, ghrelin protein appeared to be localized to the cytoplasm, whereas GHS-R1A protein was found on the plasma membrane. Within the reproductive tissues, ghrelin mRNA and protein were most abundantly expressed in the ampulla of the oviduct. Concentrations of GHS-R1A were lower than those of ghrelin but differed between tissues. This is one of the first studies to provide molecular evidence for the presence of ghrelin and GHS-R1A within the entire reproductive tract. However, implications for fertility remain to be determined.

Effects of heat stress and nutrition on lactating Holstein cows: II. Aspects of hepatic growth hormone responsiveness.

Heat stress (HS) is a multibillion-dollar problem for the global dairy industry, and reduced milk yield is the primary contributor to this annual economic loss. Feed intake declines precipitously during HS but accounts for only about 35% of the decreased milk synthesis, indicating that the physiological mechanisms responsible for decreased milk production during HS are only partly understood. Thus, our experimental objectives were to characterize the direct effects of HS on the somatotropic axis, a primary regulator of metabolism and milk yield. We recently reported no differences in mean growth hormone (GH) concentrations, GH pulsatility characteristics, or GH response to growth hormone releasing factor in HS versus pair-fed (PF) thermoneutral controls. Despite similarities in circulating GH characteristics, plasma insulin-like growth factor (IGF)-I concentrations were reduced during heat stress conditions but not in PF animals, suggesting that uncoupling of the hepatic GH-IGF axis may occur during HS. We investigated this possibility by measuring proximal indicators of hepatic GH signaling following a GH bolus. Heat stress but not PF decreased abundance of the GH receptor and GH-dependent signal transducer and activator of transcription (STAT)-5 phosphorylation. Consistent with reduced GH signaling through STAT-5, basal hepatic IGF-I mRNA abundance was lower in HS cows. Thus, the reduced hepatic GH responsiveness (in terms of IGF-I gene expression) observed during HS appears to involve mechanisms at least partially independent of reduced nutrient intake. The physiological significance of reduced hepatic GH receptor abundance during HS is unclear at this time. Aside from reducing IGF-I production, it may reduce other GH-sensitive bioenergetic processes such as gluconeogenesis.

Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.

Heat stress is detrimental to dairy production and affects numerous variables including feed intake and milk production. It is unclear, however, whether decreased milk yield is primarily due to the associated reduction in feed intake or the cumulative effects of heat stress on feed intake, metabolism, and physiology of dairy cattle. To distinguish between direct (not mediated by feed intake) and indirect (mediated by feed intake) effects of heat stress on physiological and metabolic indices, Holstein cows (n = 6) housed in thermal neutral conditions were pair-fed (PF) to match the nutrient intake of heat-stressed cows (HS; n = 6). All cows were subjected to 2 experimental periods: 1) thermal neutral and ad libitum intake for 9 d (P1) and 2) HS or PF for 9 d (P2). Heat-stress conditions were cyclical with daily temperatures ranging from 29.7 to 39.2 degrees C. During P1 and P2 all cows received i.v. challenges of epinephrine (d 6 of each period), and growth hormone releasing factor (GRF; d 7 of each period), and had circulating somatotropin (ST) profiles characterized (every 15 min for 6 h on d 8 of each period). During P2, HS cows were hyperthermic for the entire day and peak differences in rectal temperatures and respiration rates occurred in the afternoon (38.7 to 40.2 degrees C and 46 to 82 breaths/min, respectively). Heat stress decreased dry matter intake by greater than 35% and, by design, PF cows had similar reduced intakes. Heat stress and PF decreased milk yield, although the pattern and magnitude (40 and 21%, respectively) differed between treatments. The reduction in dry matter intake caused by HS accounted for only approximately 35% of the decrease in milk production. Both HS and PF cows entered into negative energy balance, but only PF cows had increased (approximately 120%) basal nonesterified fatty acid (NEFA) concentrations. Both PF and HS cows had decreased (7%) plasma glucose levels. The NEFA response to epinephrine did not differ between treatments but was increased (greater than 50%) in all cows during P2. During P2, HS (but not PF) cows had a modest reduction (16%) in plasma insulin-like growth factor-I. Neither treatment nor period had an effect on the ST response to GRF and there was little or no treatment effect on mean ST levels or pulsatility characteristics, but both HS and PF cows had reduced mean ST concentrations during P2. In summary, reduced nutrient intake accounted for just 35% of the HS-induced decrease in milk yield, and modest changes in the somatotropic axis may have contributed to a portion of the remainder. Differences in basal NEFA between PF and HS cows suggest a shift in postabsorptive metabolism and nutrient partitioning that may explain the additional reduction in milk yield in cows experiencing a thermal load.

Effects of a supplemental yeast culture on heat-stressed lactating Holstein cows.

Multiparous, lactating Holstein cows (n = 23; 120 +/- 30 d in milk, 690 +/- 67 kg of body weight) housed in climatic chambers were randomly assigned to 1 of 2 dietary treatments: a diet containing a novel yeast culture formulation (YC) for heat stress (n = 12, 10 g/d) or a control diet (n = 11). The trial length was 28 d and consisted of a 7-d thermal neutral period (TN; 18 degrees C, 20% humidity) followed by 21 d of heat stress (HS; cyclical daily temperatures ranging from 29.4 to 37.8 degrees C and 20% humidity). Cows were individually fed a total mixed ration consisting primarily of alfalfa hay and steam-flaked corn. During TN, the YC feeding had no effect on production variables or most body temperature indices. During HS, all body temperature indices increased and YC had no effect on rump surface temperature, respiration rate, or sweating rates. Cows fed YC had lower rectal temperatures at 1200 and 1800 h (40.29 vs. 40.02 degrees C and 40.35 vs. 40.12 +/- 0.07 degrees C, respectively) compared with control-fed cows. Cows fed both diets lost body weight (42 kg) during HS, but there were no differences between diets. Control-fed cows had increased dry matter intake (DMI) and milk yield (19.1 vs. 17.9 +/- 0.5 kg/d and 32.15 vs. 29.15 +/- 0.02 kg/d, respectively) compared with YC-fed cows, but intake and milk production were similar between diets when evaluated on a body weight basis. Heat stress progressively decreased DMI (29%) and milk yield, with milk production reaching a nadir (33%) in the third week. Heat stress decreased milk protein (7%) and lactose (5%) levels, but did not alter milk fat content. Heat-stressed cows were in calculated negative energy balance (-1.91 +/- 0.70 Mcal/d) and this was unaffected by diet. Independent of diet, HS decreased plasma glucose (11%), but neither diet nor HS altered basal nonesterified fatty acid levels. Heat stress increased plasma urea N concentrations (11.5 vs. 14.8 +/- 0.4 mg/dL). Despite YC-fed cows having slightly reduced body temperatures indices, feeding YC did not prevent the negative effects of HS.

Growth hormone receptor, insulin-like growth factor (IGF)-1, and IGF-binding protein-2 expression in the reproductive tissues of early postpartum dairy cows.

The growth hormone/insulin-like growth factor (IGF) system plays a critical endocrine role controlling nutrient metabolism in dairy cattle. In liver, growth hormone receptor (GHR) and IGF-1 are dynamically regulated by lactation and energy balance. Less is known about the regulation of GHR, IGF-1, and IGF-binding protein mRNA in reproductive tissues (uterus, ovarian follicle, and corpus luteum). The objective was to determine expression patterns for GHR, IGF-1, and IGF-binding protein (IGFBP)-2 mRNA in the liver, uterus, dominant follicle, and corpus luteum in Holstein cows (n = 21) sampled at 3 times during early lactation. The first postpartum ovulation was induced with an injection of GnRH within 15 d of calving. Nine days after ovulation [23 +/- 1 d postpartum; 20 d in milk (DIM)], the liver, uterus, dominant follicle, and corpus luteum were biopsied. Prostaglandin F(2alpha) and GnRH were injected 7 and 9 d after each biopsy to synchronize the second (41 +/- 1 d postpartum; 40 DIM) and third (60 +/- 1 d postpartum; 60 DIM) tissue collections. Total RNA was isolated and used for mRNA analysis by real-time quantitative reverse transcription PCR. Liver had more GHR, IGF-1, and IGFBP-2 mRNA than the reproductive tissues that were tested. Gene expression for GHR, IGF-1, and IGFPB-2 within tissues did not change across the sampling interval (20 to 60 DIM). The only detected change in gene expression across days was for cyclophilin in uterus (increased after 20 DIM). Parity had an effect on gene expression for GHR in corpus luteum. Neither level of milk production nor body condition score affected the amount of GHR, IGF-1, or IGFBP-2 mRNA in the respective tissues. The repeatability of gene expression within a tissue was 0.25 to 0.5 for most genes. In most instances, expression of a single gene within a tissue was correlated with other genes in the same tissue but was not correlated with the same gene in a different tissue. We did not find evidence for major changes in gene expression within reproductive tissues in postpartum cows. Differences between cows (independent of their BCS and milk production) accounted for a major portion of the variation that we observed.

Uterine and hepatic gene expression in relation to days postpartum, estrus, and pregnancy in postpartum dairy cows.

The somatotropic axis consisting of growth hormone, the growth hormone receptor (GHR) insulin-like growth factor (IGF)-I, and IGF binding proteins changes with the stage of lactation and nutrition of the cow and may be 1 mechanism through which lactation and nutrition affect the establishment of pregnancy. The objective of this study was to quantify GHR, IGF-I, and IGF binding protein-2 (IGFBP-2) mRNA in liver and uterine endometrial tissue at 4 stages of lactation (40, 80, 120, and 160 days in milk) and around the time of artificial insemination. Estrus was synchronized with GnRH and PGF2alpha, and cows were inseminated 12 h after estrus. Uterine biopsies were collected immediately before the second injection of PGF2alpha (before estrus), at the initiation of standing estrus, and 4 d after estrus. Liver biopsies were collected once on 4 d after estrus. The abundance of GHR, IGF-I, and IGFBP-2 mRNA in liver and uterus was determined by real-time quantitative PCR. The amount of liver IGF-I mRNA was positively correlated with plasma IGF-I concentrations. Cows that became pregnant after AI had more GHR and IGFBP-2 mRNA in their liver than cows that did not become pregnant. There was no effect of DIM or pregnancy status on abundance of uterine mRNA; however, uterine GHR and IGF-I mRNA was most abundant at estrus. In summary, cows at different stages of lactation or with different pregnancy statuses had similar quantities of uterine mRNA. In contrast, liver quantities of mRNA differed relative to pregnancy status. These data provide evidence that liver indices of metabolic state may be indicative of pregnancy success.

Timed artificial insemination of two consecutive services in dairy cows using prostaglandin F2alpha and gonadotropin-releasing hormone.

Timed artificial insemination (TAI) protocols use PGF(2alpha) and GnRH injections to synchronize ovulation. The objective was to evaluate the PGPG protocol (d 0, PGF(2alpha); d 3, GnRH; d 11, PGF(2alpha); d 13, GnRH and TAI) for first TAI and also examine methods for second TAI in nonpregnant cows. A factorial test of the first PGF(2alpha) and first GnRH injections within the PGPG protocol was performed (the last PGF(2alpha) and GnRH injections were deemed essential to the TAI). Lactating dairy cows (n = 804) in a commercial herd were assigned to 1 of 5 first-TAI treatments, which were PGPG, GPG (d 0, no treatment; d 3, GnRH; d 11, PGF(2alpha); d 13, GnRH and TAI), PPG (d 0, PGF(2alpha); d 3, no treatment; d 11, PGF(2alpha); d 13, GnRH and TAI), and PG (d 0, no treatment; d 3, no treatment; d 11, PGF(2alpha); d 13, GnRH and TAI); the Ovsynch protocol (GnRH, 7 d, PGF(2alpha), 2 d, GnRH and TAI) was the positive control. For resynchronization, cows received either GnRH or the control (no injection) on d 22 after TAI. Nonpregnant cows on d 28 were then treated with PGF(2alpha) on d 29, GnRH on d 31, and TAI [i.e., resynchronization treatments of ReGPG (received GnRH on d 22) and RePG (did not receive GnRH on d 22)]. Pregnancy rates for PGPG, GPG, PPG, PG, and Ovsynch were similar at d 28 after first TAI. Analyses of multiple explanatory factors by logistic regression detected an effect of uterine or ovarian abnormality on the d-28 pregnancy rate (normal more likely to be pregnant). Day-42 pregnancy rates were affected by uterine or ovarian abnormality (normal more likely to be pregnant), postpartum disease occurrence (healthy cows more likely to be pregnant), milk production, and days in milk. Treatment was not significant for the d-42 pregnancy rate. Effects of postpartum disease, milk production, and days in milk on the d-42 pregnancy rate were apparently manifested through their effects on embryonic loss between d 28 and 42 of pregnancy. High-producing cows that received TAI early postpartum were most likely to experience embryonic loss. Day-42 pregnancy rates after the resynchronization treatment were affected by an interaction of the first synchronization treatment with the resynchronization treatment. We concluded that although PGPG can be used for TAI, a simpler TAI protocol that includes the last 2 injections (PGF(2alpha), 2 d; GnRH and TAI) would be equally effective.