Physiology and Endocrinology Symposium
Awardee Talk: In Pursuit of Novel Factors that Regulate Ovarian Follicular Function – Dr. Leon Spicer, Oklahoma State University
The Physiology and Endocrinology Symposium on Thursday, July 15th, began with the presentation of the Animal Physiology and Endocrinology Award to Dr. Leon Spicer, recognizing him for his research that has contributed to our understanding of intra-ovarian control of ovarian function in farm animals. Congratulations, Dr. Spicer! Dr. Spicer began by discussing how reproductive efficiency is one of the main factors affecting profitability in farm animals and how the overall goal is to improve reproductive efficiency. Understanding the mechanisms that control ovarian follicular development may lead to ways to improve reproductive efficiency. Dr. Spicer continued by summarizing a few studies that utilized microarray analysis to discover various unique genes involved in ovarian follicular development
In the first study, he characterized porcine granulosa cell gene expression in response to being cultured with IGF-I. There were many genes that were differentially expressed by IGF-1. Dr. Spicer specifically discussed thrombospondin 1, an inhibitor of angiogenesis, which was downregulated and fibroblast growth factor receptor 2IIIc, the main receptor for FGF2 and FGF9, which was upregulated. The second study compared bovine granulosa cell gene expression in bovine cystic versus normal follicles to potentially aid in understanding additional causes of anovulation and ovarian follicular cyst formation. A few novel genes discovered included Indian hedgehog protein (IHH), FGF9, brain ribonuclease (BRB), and GPR34. Overall, BRB, GPR34, WNT, IHH, and IGF-1 may regulate mitogenic and angiogenic factors produced by the follicle while stimulating FSH and LH-induced differentiation. On the other hand, FGF9, GDF9, and BMP4 may induce mitosis and inhibit steroidogenesis of theca and granulosa cells. The vascular network that surrounds the follicle is key in the developmental process and is very intricate and complex with many factors involved. Dr. Spicer concluded by stating it is important to see if any of these identified genes can be manipulated to potentially reduce the number of follicles that undergo atresia, improve the quality of follicles, prevent cystic follicle formation, and maybe ultimately improve fertility of our animals and even oocyte quality.
W112 Reproductive Performance in Domestic Ruminants: 50 years of accomplishment: A USDA-NIFA Multistate Research Project – Dr. Milan Shipka, University of Alaska Fairbanks
Dr. Shipka began his presentation by acknowledging the founders of W112 Reproductive Performance in Domestic Ruminants, which was preceded by W49 studying reproduction and that group preceded by W2 back in 1947. The beginning of this multistate group, therefore, goes more than 50 years back. The W2 group was changed to W49 in 1956, focusing on physiological causes of breeding failure in cattle, and then in 1969 the multistate project title of Reproductive Performance in Domestic Ruminants was proposed and approved in 1970, marking the start of W112. Dr. Shipka continued on by discussing work completed by this multistate project over the years such as looking at estrous cycle and assay development to ovarian function, ovulation, CL formation, and parturition to estrous synchronization, embryo transfer, nutrition and reproduction, maternal recognition of pregnancy, and bull fertility to developing comparative physiology and model development, and finally in the last 20 years to looking more at environmental influences on reproduction, maternal recognition of pregnancy, fetal development and maturation, pregnancy maintenance, fetal programming, ad early embryonic loss.
More and more stations became involved in W112 as productivity of the group increased and people were recognizing of the impacts of the group and quality of the scientists participating. Dr. Shipka stressed that while this group was of great importance from a research point of view, they also went beyond research and were invaluable as far as outreach and mentoring. This group put an effort into taking the knowledge they generated and make sure it reached other scientists, extensionists and producers by putting together handbooks and setting up symposia to try and magnify their messages. Project participants also share teaching resources, share graduate students, advising on career and advancement, recruitment for faculty positions, overall fostering future involvement in the project. The W112 Reproductive Performance in Domestic Ruminants has produced more than 50 years of advancement in our understanding of reproductive biology in ruminant animals while creating a group of people that are a group of friends, colleagues, collaborators, advisors, mentors, supporters, and still have much more to contribute as it moves into the next 50 years.
Effects of Inflammatory States on Ovarian Biology and Oocyte Competence – Dr. Jennifer Hernandez Gifford, New Mexico State University
Dr. Gifford continued the discussion focusing on the importance of estrogen in female fertility and how the synthesis of estrogen relies on coordinated actions of FSH, IGF-1, and other intra-ovarian signaling molecules and control mechanisms. Anything that disrupts estrogen regulation can disrupt folliculogenesis and oocyte competency and may lead to female infertility. One specific control mechanism Dr. Gifford highlighted was the co-transcription factor beta-catenin, which is a multi-functional protein that has a role in granulosa cell steroid production. Beta-catenin is regulated by FSH in bovine granulosa cells that then contributes to the overall regulation of aromatase gene expression that consequently correlates to estradiol output from that follicle.
Dr. Gifford then went on to explain a new area of research looking at impacts of disease on fertility in the female, specifically focusing on bacterial infections caused by pathogenic gram-negative bacteria that produce LPS, an endotoxin responsible for stimulation of the immune system. The ability of this endotoxin to disrupt reproduction is coming into light now as the severity on reproductive issues, even in an asymptomatic infection, is very real and can lead to overall infertility of an animal. She discussed how LPS is detrimental to aromatase and estradiol production in dairy cattle. Beta-catenin has been shown to be modulated by LPS and itself be a modulation of inflammatory responses such as regulating expression of various inflammatory cytokines in cells with LPS. Overall, induced or naturally occurring LPS, even at low doses, has been shown to modulate the intra-follicular hormonal milieu including circulating and follicular estradiol concentrations that may impair oocyte maturation. Inflammatory disease, especially subclinical disease, needs to be researched more as a factor that alters fertility, leading to decreased cow retention.
Reproductive Microbiomes as Predictors of Fertility in Beef Cattle – Dr. Rebecca Poole, Texas A&M University
Dr. Poole began her presentation by discussing the complexity of the reproductive microbiome and its role as it pertains to fertility. Comparing the vaginal microbiome of healthy women to the vaginal microbiome of cattle showed significant differences. In healthy women, the vaginal microbiome predominantly consists of bacteria in the genus Lactobacillus, which lowers the vaginal pH and protects against pathogens that may be introduced into the reproductive tract to hopefully improve fertility outcomes. On the other hand, in the vaginal microbiome of cattle there are low abundances of Lactobacillus, a much greater diversity of bacterial species present (alpha-diversity), and a more neutral pH around 7.3 as compared to a human vaginal pH < 4.5.
Dr. Poole then analyzed the differences in bacterial species in the vaginal and uterine microbiomes in cattle between resulting pregnant and non-pregnant cows and evaluate the overall change over the duration of the synchronization protocol. Beta-diversity analysis showed there was a shift in the relative abundances of bacteria in the uterus leading up to timed artificial insemination (TAI) with the resulting pregnant cows having greater variation, potentially suggesting that there may not be one healthy uterine microbiome to support pregnancy, but rather a closely related group of bacteria that negatively impact fertility. Dr. Poole also discussed the immune system in relation to the microbiome, stating that changes in uterine cytokine concentrations, specifically a reduction in uterine TGF-beta and greater uterine IL-6, appear to be associated with an abundance of pathogenic bacteria prior to TAI in resulting non-pregnant beef cows. Overall, the reproductive microbiome is very complex and seems to be altered by various mechanisms that in turn may impact fertility.
Characterizing Vaginal Bacterial Community Composition in Brangus Heifers – Dr. Caleb Lemley, Mississippi State University
Dr. Lemley continued the symposium by further discussing the vaginal microbiota in relation to fertility, conception, pregnancy, and parturition, again highlighting the difference between the acidic vaginal microbiota dominated by Lactobacillus in humans and the neutral, dynamic vaginal microbiota in cattle. He stressed that the vaginal microbiome has many potential implications such influencing acquisition of venereal pathogens, reproductive outcomes, and perinatal health and calf performance due to early contact of the neonate with microorganisms during parturition. Studies analyzing the bovine vaginal microbiota during the estrous cycle, gestation, and postpartum period have been performed, determining that the bovine vaginal microbiota is dynamic.
Other studies started to investigate ways we could alter the composition of the bovine vaginal microbiota such as with dietary melatonin supplementation, endogenous progesterone, maternal nutrient restriction, and gestational age. Dr. Lemley discussed that maternal nutrient restriction did not alter alpha or beta diversity of the vaginal microbiota and dietary melatonin supplementation did not alter alpha diversity of the vaginal microbiota but did alter beta diversity of the vaginal microbiota increasing the prevalence of aerobic bacteria in the vaginal tract. More research needs to be conducted evaluating the role of the reproductive microbiome to potentially discover mechanisms that could be altered to increase reproductive performance.
The Role of Extracellular Vesicles in Mitigating the Effect of Heat Stress – Dr. Dawit Tesfaye, Colorado State University
Heat stress is an environmental stressor that negatively impacts the fertility of dairy cows by disrupting reproductive processes starting with follicular development all the way to maternal recognition of pregnancy. Dr. Tesfaye’s presentation focused on the role of extracellular vesicles (EVs) in mitigating the effect of heat stress. He discussed how EVs are produced by many cell types and are present in various biological fluids including uterine fluids in vivo, spent culture media in vitro, follicular fluid, and oviductal fluid. They contain biologically active signaling molecules, such as proteins, lipids, miRNA, mRNA, that have been shown to have an important role in the cellular response to stress.
Dr. Tesfaye continued by explaining the importance of EVs as molecular cargo in biological fluids being an indicator of the physiological status of the animal, such as the presence of EV-mediated miRNA signals in follicular fluid that was associated with the overall metabolic status of cows. There may be a potential role of follicular fluid EVs in carrying molecular signals that may protect or even reverse damage incurred by heat stress in bovine oocytes. By understanding how these EVs are enriched with specific mRNA and miRNAs and contribute to cell-to-cell communication with the delivery of these signals, we could potentially utilize these EVs to combat oxidative stress-associated fertility problems.
Physiological Responses to Heat Stress of Holstein Heifers Carrying the SLICK1 Allele of the Prolactin Receptor (PRLR) Gene – Dr. Anna Denicol, University of California Davis
Dr. Denicol continued with the discussion on heat stress, saying it is one of the main causes of economic losses in the dairy industry. Rather than looking at reproduction directly, she wants to try to find long term solutions to heat stress, which remains a big barrier to reproductive efficiency in cattle. While there are effective management strategies in place to try to minimize heat stress, they increase production costs and water usage. Dr. Denicol turned the focus onto genetic selection for thermotolerance as potentially a permanent or at least partial solution to ameliorate the detrimental effects of heat stress on production, reproduction, and welfare of dairy cows.
She specifically evaluated the physiological responses to heat stress in Holstein heifers who inherited the SLICK1 allele of the prolactin receptor gene (PRLR), a mutation that results in a phenotype of sleek, shorter hair coat, in California and Florida where these heifers are subject to heat stress for longer periods of time. Slick heifers in Florida had lower rectal temperatures compared with non-slick siblings, but this difference was not seen in California. In both state, slick heifers tended to have lower surface temperatures. California heifers had lower rectal temperatures and surface temperatures and higher sweating rates compared to the Florida heifers. Dr. Denicol stressed that an important question would be if the introduction of this SLICK1 allele negatively impacts fertility, but so far she reported the heifers carrying the allele had similar fertility to their non-slick half siblings and unrelated cohort animals. Moving forward, she will continue to monitor these animals as they calf themselves to evaluate any effects on milk yield and composition.
Effects of Dam Nutrition on Offspring Metabolism – Dr. Kristen Govoni, University of Connecticut
After the lunch break, Dr. Govoni continued the symposium shifting from a reproductive focus of the previous presenters to the fetal programming area and trying to find ways to improve efficiency of production. Fetal programming is a concept that suggests changes in the intrauterine/maternal environment during gestation may have long-term impacts on the offspring. Maternal diet during gestation is important for proper fetal growth and development and evidence shows that poor maternal nutrition, consisting of restricted- and over-feeding, can alter the growth of the fetus with long-term consequences on postnatal growth and development and eventually, adult maintenance.
Dr. Govoni discussed how a suboptimal maternal-fetal nutritional environment during gestation can ultimately program the offspring for altered metabolism, such as increased adipose deposition, hyperinsulinism, cardiovascular disease, leptin resistance, all of which can likely reduce efficiency of production later in life. There is evidence that poor maternal nutrition also alters key metabolic profiles in muscle and liver in the offspring. There are various mechanisms that may be behind offspring metabolic dysregulation such as epigenetic regulation, altered stem cell population and function, altered gene and protein expression of key factors in metabolism, and changes in key metabolites, that are ultimately contributing to the long-term reduction in efficiency of growth and product quality.
Effects of Transition Period Nutrition on Uterine Health – Dr. Felipe Cardoso, University of Illinois
Dr. Cardoso started his discussion by highlighting the importance of understanding that livestock species have different limiting nutrients depending on their main output and that some effects we see in sheep or cattle will be different based on that fact. He then explained that any study that is done should try to address exactly what we want from a cow: to minimize health disorders, maximize production of milk, and maximize reproduction. The transition period, the transition from gestation to lactation, is a very critical time for dairy cows. Formulating and delivering an appropriate diet that limits total energy intake to requirements while providing proper intakes of all other nutrients before calving may help lessen the extent of negative energy balance (NEB) after calving, potentially decreasing disease incidence and increasing capability of both high milk yield and successful reproduction.
Reproduction is affected by events occurring earlier in lactation, bringing us back to the transition period, as well as a proper uterine environment, both of which we can manipulate through nutrition. Dr. Cardoso then discussed some diets that could be given to cows during the transition period to help improve uterine health and ultimately fertility in dairy cows such as a controlled-energy diet during the last 3 weeks prepartum, a fully acidified prepartum diet with higher Ca concentration, and supplementation of rumen-protected methionine. Moving forward, it is important to look at specific moments throughout lactation as the cow is metabolically going through different processes.
Effect of Dam Nutrition on Offspring Reproductive Performance – Dr. John Hall, University of Idaho
Dr. Hall examined the effects of maternal undernutrition on subsequent reproductive performance of female offspring with an emphasis on beef cattle. He discussed how domestic ruminants rely heavily on grazed forages as their primary source of nutrition and how these forage sources vary greatly in nutrient content and availability throughout the year. This causes the ruminants to be exposed to nutrient excesses and deficiencies at various time points throughout their reproductive cycle. There is limited research on the effect of undernutrition during early gestation in cattle and many studies do not go beyond fetal development or assess subsequent fertility based on indicators of fertility such as anti-mullerian hormone (AMH) and antral follicle count (AFC).
When looking at restriction in early gestation, severe nutrient deprivation of heifers led to female offspring having reduced AMG and AFC, while a more moderate nutritional challenge did not produce changes in AMH and AFC in female offspring. Dr. Hall also stated that there were no differences found in age of puberty. When looking at restriction in late gestation, heifers from dams that grazed protein deficient ranges had delayed puberty onset and decreased pregnancy rates compared with heifers from supplemented dams. A limited number of studies have quantified effects on fertility due to nutrient restriction or supplementation warranting more investigations of maternal nutrition impacts on reproduction in offspring.
Effects of Nutrition on Bull Fertility – Dr. Tom Geary, USDA-ARS, Fort Keogh ARRL
Dr. Geary shifted the focus to examining the effects of nutrition on bull fertility during the prepubertal phase and the peripubertal phase. Increasing the plane of nutrition during the prepubertal phase (< 6 months) lead to a decrease in age of puberty, increased scrotal size, and increased total sperm production. Dr. Geary explained that we can manipulate the diet during this phase by creep feeding bull calves with protein and energy supplements to ultimately get that accelerated puberty and increase in sperm production, but we have to be careful doing this because increasing the plane of nutrition during the peripubertal phase (6 to 16 months) can lead to an increase in weight at puberty and an increase in scrotal fat that can potentially cause thermoregulation problems resulting in decreased semen quality and detrimental effects on fertility. In contrast, decreasing the plane of nutrition for mature bulls during the off-season actually revealed enhanced sperm energy and stress-fighting potential. Dr. Geary concluded that if anything, over-feeding seems to produce greater negative effects in terms of fertility.
Trace mineral supplementation, such as with copper and zinc, has seen to be critical for optimal male fertility in other species, so Dr. Geary examined the effects of minerals on bull fertility. Providing these minerals to peripubertal bulls resulted in increased liver concentrations of mineral and in some cases, accelerated puberty, but overall, enhanced fertility-associated measures were not realized. Perhaps this finding would be different in bulls during the breeding season since copper and zinc are present in the ejaculate. Moving forward there are some unanswered questions that need to be analyzed such as what are specific nutrient requirements of beef bull, do they vary by breeding season and what are the nutritional effects during breeding season.
Awardee Talk: Maternal versus Paternal Contributions to Fertility – Dr. Ky Pohler, Texas A&M University
After returning from a short break, Dr. Pohler was presented with the Early Career Achievement Award. Congratulations, Dr. Pohler! He then continued the symposium by addressing the idea that reproductive success is highly variable and is influenced by both maternal and paternal factors. Aside from the numerous maternal factors involved with pregnancy establishment such as reproductive tract size, estrus expression, endocrine environment, the sire also makes contributions to reproductive success and failure that should not be overlooked. When examining differences in sire field fertility, Dr. Pohler discussed how there were no differences in semen characteristics nor in sire conception rate to explain the differences in sire field fertility during the second month of gestation in beef and dairy herds.
To get a better understanding of the maternal and paternal contributions to pregnancy development during the second month of gestation specifically, Dr. Pohler explained how he developed parthenogenetic embryos, embryos that lack a paternal genome. While the parthenogenetic embryos had well developed trophectoderm tissue during gestation, there was no site of embryo attachment to the endometrium and pregnancy-associated glycoproteins and interferon-stimulated genes were not found in maternal circulation suggesting the important role of paternal genetics in successful post-elongated embryo attachment to endometrium. More research needs to be conducted analyzing maternal versus paternal contribution to fertility to potentially help clarify what leads to decreased fertility and reproductive failure.
Using in vivo RNA Interference to Investigate Ruminant Placental Function – Dr. Russell Anthony, Colorado State University
Dr. Russell began his discussion by stating while it is important to get a calf on the ground, what is more important is getting that calf to reach its genetic capacity ex utero. There are many reasons why this does not happen all the time and many places where processes can go wrong. He then explained how the placenta is the master organ of the body as it can do almost anything the rest of the body can do. While our understanding of the in vivo physiology of pregnancy in sheep is unmatched by any other species, there has been a significant deficit in determining specific functions of individual genes expressed by the placenta in livestock. Dr. Russell described how lentiviral-mediated in vivo RNA interference within the sheep placenta is a useful approach to investigate the function of specific gene products involved in conceptus development (PRR15), placental nutrient transport (SLC2A3), and placenta derived hormones (CSH) by knocking down gene expression.
Having the ability to place indwelling catheters within both maternal and fetal vessels, allows for the use of the transplacental diffusion technique along with the Fick Principle to assess uterine and umbilical blood flows, uterine and umbilical nutrient uptakes, and placental utilization. This removes the need for anesthesia and creates non-stressed steady-state conditions that will allow us to really understand what is going on in the in-utero environment and get a direct assessment of any changes in that environment that may tie to postnatal growth and production traits as previously discussed today.
W4112 Reproductive Performance in Domestic Ruminants: The next 50 years – A USDA-NIFA Multistate Research Project – Dr. Thomas Hansen, Colorado State University
Dr. Hansen wrapped up the symposium discussing the objectives of the W4112 Reproductive Performance in Domestic Ruminants and where the beef and dairy cattle industries would be in 50 years. A few of the objectives he mentioned include clarifying mechanisms that regulate gamete development and quality, identifying impacts of reproductive management, animal management, and stress on follicle recruitment, ovulation, and pregnancy, determining mechanisms that regulate normal embryo development and pregnancy establishment, and exploring maternal and paternal factors.
Based on the expected world population growth, Dr. Hansen concluded that there will be a continued need to feed the growing human population high quality animal protein. The animal protein may be replaced over time by meat substitutes, which currently has its own little market share, but the beef demand will likely continue to trend upward as it has recently. He then went on to discuss the impact of cattle on the environment, why animal health matters for our environment, the impact of poor reproduction on ruminant production systems, future implementation of technologies, and genome modifications that give rise to many interesting state of the art opportunities. Dr. Hansen highlighted the major impacts W112 had over the years such as our ability to produce the same amount of beef with a decreasing cattle inventory having a smaller impact on environment and similar with milk production skyrocketing while dairy cow inventory is decreasing. Overall, there have been large gains in productivity and other very respectable, efficient progress that is expected to continue within the beef and dairy cow industries as well as other ruminant species over the next 50 years.