Regulatory mechanisms for natriuretic peptide signalling in sheep granulosa cells.

Natriuretic peptides (NPs) have been reported to have critical roles in follicular development and oocyte maturation in rodents. This study aimed to extend our current understanding of NP-mediated signalling pathways and mechanisms of action in the follicles of a monovulatory species. Ovine granulosa cells (GCs) and theca cells (TCs) were cultured under conditions designed to allow gonadotrophin-stimulated cell differentiation. Gene expression analysis was performed by qualitative (q)PCR for NPs and NPRs (between 16 and 96 h of culture) and VEGF120 and VEGF164 (between 16 and 144 h of culture). A qualitative analysis of the production of NP/NPR family members and NP ligand/receptor associations was carried out utilising a highly sensitive immunological approach known as 'proximity ligation assay' (PLA). All NPRs were observed in GCs, while NPRA was absent in TCs. In GCs, gene expression of NPRA, NPRB and NPRC was apparent but only active BNP and CNP and not ANP, were detected. Also in GCs, ANP but not CNP was able to significantly (P < 0.05) reduce oestradiol and increase (P < 0.05) progesterone. Inhibition of VEGF164 by ANP and CNP (P < 0.01) after 48 h of culture preceded up-regulation of VEGF120 by ANP (P < 0.01) after 144 h, but not CNP. Taken together, these findings appear to demonstrate that NP responsiveness in the GC compartment of sheep follicles is multi-facilitated, utilising both autocrine and paracrine stimulation pathways.

Maternal periconceptional and first trimester protein restriction in beef heifers: effects on placental parameters and fetal and neonatal calf development.

Few studies have investigated the effects of nutrition during the periconception and early gestation periods on fetal and placental development in cattle. In this study, nulliparous yearling heifers (n=360) were individually fed a diet high or low in protein (HPeri and LPeri) beginning 60 days before conception. From 24 to 98 days after conception, half of each treatment group was changed to the alternative high- or low-protein diet (HPost and LPost) yielding four groups in a 2×2 factorial design. A subset of heifers (n=46) was necropsied at 98 days after conception and fetoplacental development assessed. Placentome number and volume decreased in response to LPeri and LPost diets respectively. Absolute lung, pancreas, septum and ventricle weights decreased in LPost versus HPost fetuses, whereas the post-conception diet altered absolute and relative liver and brain weights depending on sex. Similarly, changes in fetal hepatic gene expression of factors regulating growth, glucose output and lipid metabolism were induced by protein restriction in a sex-specific manner. At term, neonatal calf and placental measures were not different. Protein restriction of heifers during the periconception and early gestation periods alters fetoplacental development and hepatic gene expression. These changes may contribute to functional consequences for progeny, but this may not be apparent from gross morphometry at birth.

Genome-Wide Association Studies for Methane Production in Dairy Cattle.

Genomic selection has been proposed for the mitigation of methane (CH4) emissions by cattle because there is considerable variability in CH4 emissions between individuals fed on the same diet. The genome-wide association study (GWAS) represents an important tool for the detection of candidate genes, haplotypes or single nucleotide polymorphisms (SNP) markers related to characteristics of economic interest. The present study included information for 280 cows in three dairy production systems in Mexico: 1) Dual Purpose (n = 100), 2) Specialized Tropical Dairy (n = 76), 3) Familiar Production System (n = 104). Concentrations of CH4 in a breath of individual cows at the time of milking (MEIm) were estimated through a system of infrared sensors. After quality control analyses, 21,958 SNPs were included. Associations of markers were made using a linear regression model, corrected with principal component analyses. In total, 46 SNPs were identified as significant for CH4 production. Several SNPs associated with CH4 production were found at regions previously described for quantitative trait loci of composition characteristics of meat, milk fatty acids and characteristics related to feed intake. It was concluded that the SNPs identified could be used in genomic selection programs in developing countries and combined with other datasets for global selection.

The Effects of Developmental Programming upon Neonatal Mortality.

The greatest loss in ruminant production systems occurs during the neonatal period. The maternal environment (nutrition and physiologic status) influences neonatal mortality and morbidity as it reportedly affects (a) Dystocia, both via increasing birth weight and placental dysfunction; (b) Neonatal thermoregulation, both via altering the amount of brown adipose tissue and its ability to function via effects upon the hypothalamic-pituitary-thyroid axis; (c) Modification of the developing immune system and its symbiotic nutrient sources; (d) Modification of maternal and neonatal behavior.

Subacute ruminal acidosis reduces sperm quality in beef bulls.

Breeding bulls are commonly fed high-energy diets, which may induce subacute ruminal acidosis (SARA). In this experiment, 8 Santa Gertrudis bulls (age 20 ± 6 mo) were used to evaluate the extent and duration of effects of SARA on semen quality and the associated changes in circulating hormones and metabolites. The bulls were relocated and fed in yards with unrestricted access to hay and daily individual concentrate feeding for 125 d before SARA challenge. Semen was collected and assessed at 14-d intervals before the challenge to ensure acclimatization and the attainment of a stable spermiogram. The challenge treatments consisted of either a single oral dose of oligofructose (OFF; 6.5 g/kg BW) or an equivalent sham dose of water (Control). Locomotion, behavior, respiratory rate, and cardiovascular and gastrointestinal function were intensively monitored during the 24-h challenge period. Rumen fluid samples were retained for VFA, ammonia, and lactate analysis. After the challenge, semen was then collected every third day for a period of 7 wk and then once weekly until 12 wk, with associated blood collection for FSH, testosterone, inhibin, and cortisol assay. Percent normal sperm decreased in bulls dosed with OFF after the challenge period ( < 0.05) and continued to remain lower on completion of the study at 88 d after challenge. There was a corresponding increase in sperm defects commencing from 16 d after challenge. These included proximal cytoplasmic droplets ( < 0.001), distal reflex midpieces ( = 0.01), and vacuole and teratoid heads ( < 0.001). Changes in semen quality after challenge were associated with lower serum testosterone ( < 0.001) and FSH ( < 0.05). Serum cortisol in OFF bulls tended to be greater ( = 0.07) at 7 d after challenge. This study shows that SARA challenge causes a reduction in sperm quality sufficient to preclude bulls from sale as single sire breeding animals 3 mo after the event occurred.

Maternal endocrine adaptation throughout pregnancy to nutrient manipulation: consequences for sexually dimorphic programming of thyroid hormones and development of their progeny.

Maternal nutrient restriction during critical windows of fetal development alters postnatal growth, often in a sexually dimorphic manner. Intrauterine growth restriction is frequently characterized by accelerated growth and increased adiposity in later life. Thyroid hormones are implicated as part of the mechanism involved in this scenario via their actions within the hypothalamic-pituitary-thyroid axis. We fed high (H = 240%) and low (L = 70%) levels of recommended daily crude protein intake during the first and second trimesters of gestation to beef heifers to investigate effects to their progeny's plasma concentrations of free and total triiodothyronine (FT3 and TT3) and thyroxine (FT4 and TT4) from birth until weaning at 191 days of age (n = 68). The study design was a two-by-two factorial. For male progeny, exposure to maternal diets low in protein during the first trimester of gestation resulted in greater FT4 at birth (P < 0.05) which was subsequent to lower concentrations of leptin in maternal plasma at 271 days of gestation compared with their high-protein-exposed counterparts. These same animals went on to have greater milk intake during the latter half of the lactation period (P < 0.05) and exhibited faster rates of average daily gain (ADG) relative to birth weight during this time (P < 0.05). For all progeny, independent of sex, exposure to low-protein maternal diets during the second trimester of gestation resulted in greater FT3 relative to TT3 at birth. Because FT3 at birth and 29 days was positively associated with ADG (P < 0.05) and ADG relative to birth weight (P < 0.05), it is proposed that FT3 plays an integral role in catch-up growth in the bovine as per other species. Protein intake during the first and second trimesters of gestation has a sexually dimorphic effect on progeny plasma thyroid hormone concentrations, and these changes are associated with altered milk intake and postnatal growth pathway.

Restoration of ovarian function and natural fertility following the cryopreservation and autotransplantation of whole adult sheep ovaries.

STUDY QUESTION: Is it possible to restore ovarian function and natural fertility following the cryopreservation and autotransplantation of whole ovaries, complete with vascular pedicle, in adult females from a large monovulatory animal model species (i.e. sheep)? SUMMARY ANSWER: Full (100%) restoration of acute ovarian function and high rates of natural fertility (pregnancy rate 64%; live birth rate 29%), with multiple live births, were obtained following whole ovary cryopreservation and autotransplantation (WOCP&TP) of adult sheep ovaries utilizing optimized cryopreservation and post-operative anti-coagulant regimes. WHAT IS KNOWN ALREADY: Fertility preservation by WOCP&TP requires successful cryopreservation of both the ovary and its vascular supply. Previous work has indicated detrimental effects of WOCP&TP on the ovarian follicle population. Recent experiments suggest that these deleterious effects can be attributed to an acute loss of vascular patency due to clot formation induced by damage to ovarian arterial endothelial cells. STUDY DESIGN, SIZE, DURATION: Study 1 (2010-2011; N = 16) examined the effect of post-thaw perfusion of survival factors (angiogenic, antioxidant, anti-apoptotic; n = 7-8) and treatment with aspirin (pre-operative versus pre- and post-operative (n = 7-9)) on the restoration of ovarian function for 3 months after WOCP&TP. Study 2 (2011-2012; N = 16) examined the effect of cryoprotectant (CPA) perfusion time (10 versus 60 min; n = 16) and pre- and post-operative treatment with aspirin in combination with enoxaparine (Clexane(®); n = 8) or eptifibatide (Integrilin(®); n = 8) on ovarian function and fertility 11-23 months after WOCP&TP. PARTICIPANTS/MATERIALS, SETTING, METHODS: Both studies utilized mature, parous, Greyface ewes aged 3-6 years and weighing 50-75 kg. Restoration of ovarian function was monitored by bi-weekly blood sampling and display of behavioural oestrus. Blood samples were assayed for gonadotrophins, progesterone, anti-Müllerian Hormone and inhibin A. Fertility restoration in Study 2 was quantified by pregnancy rate after a 3 month fertile mating period and was confirmed by ultrasound, hormonal monitoring and live birth. Ovarian function was assessed at sacrifice by ovarian appearance and vascular patency (Doppler ultrasound) and by follicular histology. MAIN RESULTS AND THE ROLE OF CHANCE: In Study 1, survival factors were found to have no benefit, but the inclusion of pre-operative aspirin resulted in four ewes showing acute restoration of ovarian function within 3 weeks and a further six ewes showing partial restoration. The addition of post-operative aspirin alone had no clear benefit. In Study 2, combination of aspirin with additional post-operative anti-coagulants resulted in total acute restoration of ovarian function in 14/14 ewes within 3 weeks of WOCP&TP, with 9/14 ewes becoming pregnant and 4/14 giving birth to a total of seven normal lambs. There was no difference between anti-coagulants in terms of restoration of reproductive function and fertility. In contrast, the duration of CPA perfusion was highly significant with a 60 min perfusion resulting in ovaries of normal appearance and function with high rates of primordial follicle survival (70%) and an abundant blood supply, whereas ovaries perfused for 10 min had either resorbed completely and were vestigial (7/14) or were markedly smaller (P < 0.01). It is concluded that both the degree of CPA penetration and the maintenance of post-operative vascular patency are critical determinants of the success of WOCP&TP. LIMITATIONS, REASONS FOR CAUTION: Before application of this technology to fertility preservation patients, it will be critical to optimize the CPA perfusion time for different sized human ovaries, determine the optimum period and level of anti-coagulant therapy, and confirm the normality of offspring derived from this procedure. WIDER IMPLICATIONS OF THE FINDINGS: This technology holds promise for the preservation of fertility in women. It could also potentially be applied to the cryopreservation of other reproductive or even major organs (kidneys) where there are considerable difficulties in storing donated tissue. STUDY FUNDING/COMPETING INTERESTS: Funding was received from the Medical Research Council, University of Nottingham. The authors confirm that they have no conflict of interest in relation to this work.

Effects of dehydroepiandrosterone on in vivo ovine follicular development.

STUDY QUESTION: What are the effects of exposure of ovarian tissue to dehydroepiandrosterone (DHEA) supplementation in vivo? SUMMARY ANSWER: DHEA exposure stimulates initiation of primordial follicles and development of gonadotrophin-responsive preantral/early antral follicles possibly mediated through promoting granulosa cell proliferation and enhancing anti-Mullerian hormone (AMH) expression. WHAT IS KNOWN ALREADY?: Ovarian ageing is a cause of subfertility and is associated with poor outcomes of IVF treatment and premature menopause. A few clinical studies have shown that DHEA can improve ovarian response and increase the chances of pregnancy after IVF treatment in women with a diminished ovarian reserve (DOR) suggesting DHEA may help to overcome the effect of ovarian ageing. However, there are no data about how DHEA acts on ovarian folliculogenesis. STUDY DESIGN, SIZE AND DURATION: A cortical autograft experimental model was conducted in six female sheep aged at least 24 months. The period of DHEA treatment in the animals lasted for 10 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: All the animals were subjected to unilateral oophorectomy. Half of the ovary was fixed for histological analysis as a time-zero control. The remaining tissue was used to isolate patches of ovarian cortex which were autografted back onto the ovarian pedicle. The grafting procedure eradicated all growing follicles and synchronized early follicular development. After a 10-week treatment period with DHEA implants, the ewes were sacrificed and the graft and remaining ovary were harvested. Histological and immunohistochemistry (IHC) findings, accompanied with serum hormonal profiles were compared to determine the effect on the follicle population. MAIN RESULTS AND THE ROLE OF CHANCE: Higher proportions of the follicle population in the remaining ovary were observed to be in the antral stage after DHEA treatment. The observation coincided with an increase in the rate of primordial follicle initiation and preantral follicle development in cortical grafts and the remaining ovarian tissue, respectively. The IHC results indicated that DHEA increased the expression of both the proliferation marker (KI-67) in granulosa cells and the follicular AMH expression at the preantral and early antral follicle stages. LIMITATIONS, REASONS FOR CAUTION: The experimental design compared follicle populations before and after DHEA treatment within individual animals to allow changes over time to be detected against a background of high inter-animal variation. However, since no controls without DHEA were included, we cannot say what would have happened over time in its absence, and it is possible that other factors may have resulted in the changes in follicle development observed during the experiment. WIDER IMPLICATIONS OF THE FINDING: Our data supports the idea that DHEA might be a useful therapy to delay the effects of ovarian ageing. Therefore, it may have a role as an adjunct during IVF to improve ovarian response in women with DOR and as a treatment for premature ovarian insufficiency. STUDY FUNDING/COMPETING INTEREST(S): The research received finance support from the University of Nottingham. The authors declare no conflict of interest in this study.

Early postnatal immunisation against gonadotrophin-releasing hormone induces a high but differential immune response in heifer calves.

The aim of this study was to evaluate endocrinological and immunological effects of early postnatal immunisation against gonadotrophin-releasing hormone (GnRH) in heifer calves, as similar treatment in sheep provokes long-term immunocastration. Heifer calves were injected with either a construct of GnRH - bovine herpes virus 1 glycoprotein D (BHV1 gD; n=9) or saline (n=9) at 2, 6 and 13.5 weeks of age. Antibody (GnRH and carrier) and endocrine responses to immunisation were measured twice monthly (FSH and progesterone) or during intensive sampling regimes (LH). Early postnatal immunisation against GnRH induced a high, but variable, antibody response against both GnRH and carrier. Based on antibody responses, animals were divided into high-titre (HT, n=5) and low-titre (LT, n=4). Occurring mainly in HT, a further peak in anti-GnRH antibodies, stimulated independently of the carrier, was observed at 23 weeks of age, with antibody titres ≥ 10% binding for ≈ 9 weeks post-peak. Conversely immunisation had only temporary, reversible effects on reproductive function, not affecting age at puberty. We hypothesise that the newly generated antibody measured 10 weeks after the final immunisation resulted from antigenic stimulation and immunological memory cell activation to an endogenous GnRH release. This outcome offers an opportunity for further manipulation of reproductive function based on modulation of GnRH secretion and activity where long-term immunological memory may contribute to durable endocrine effects.

Neonatal immunisation against a novel gonadotrophin-releasing hormone construct delays the onset of gonadal growth and puberty in bull calves.

The aim of the present study was to investigate the effect of the neonatal immunisation of bull calves against a novel gonadotrophin-releasing hormone (GnRH) construct, comprised of GnRH coupled to the glycoprotein D subunit of the bovine herpes virus-1 (GnRH-BHV1 gD), on endocrine status, reproductive organ development and carcass quality. Eighteen bull calves received either GnRH construct (n=9) or saline (control; n=9) at 2, 6 and 13.5 weeks of age. Blood samples were taken to determine antibody titres against GnRH, FSH and testosterone (T) concentrations and LH pulse characteristics, with testicular circumference monitored monthly. Immunisation reduced LH pulse amplitude (P<0.05) and T concentrations (P<0.05), particularly at the peak in anti-GnRH titres after the second booster at 16 weeks of age (P<0.001), but not when titres fell. Despite antibody titres decreasing after 16 weeks, immunisation reduced testicular size between 16 to 57 weeks of age (P<0.05), provoking an 8-week delay in puberty onset, defined as testicular circumference ≥14 cm. In conclusion, neonatal immunisation induced a significant immune response against GnRH, provoking a temporary endocrine disturbance that had a long-term effect on testicular development, delaying the onset of puberty. These results support the hypothesis that a developmental window exists during testicular development, such that disturbance of the endocrine drive to the gonads during this period results in a longer-term impairment of gonadal function.

Nutritional influences on folliculogenesis.

Folliculogenesis is an intricate process that involves the proliferation and differentiation of both somatic and germ cells. This process depends on complex interactions between systemic factors such as both pituitary gonadotrophins and metabolic hormones and/or local factors produced by the ovarian somatic and germ cells, such as the IGF system and TGF-ß superfamily members. In domestic ruminants, follicular development begins during foetal life with formation of primordial follicles from the association of germ cells and pre-granulosa cells. After follicular formation, folliculogenesis begins with a primordial follicle progressing into more developed stages (i.e. primary, secondary, pre-antral and antral) in a continuous, progressive process to either ovulation or, as in most cases, to atresia. Even early stages of follicular formation and subsequent development are influenced by both internal (e.g. genotype) and/or external environmental (e.g. nutrition and season) factors. Among these external factors, nutrition is one of the most important affecting reproductive function, and this is the focus of this review, because other reviews in this issue discuss other environmental factors. A number of studies have now shown that nutrition can have both positive and negative effects on follicular growth, oestrous activity, oocyte quality, blastocyst development and pregnancy outcome. Therefore, understanding the intricate processes involved during folliculogenesis and the ways in which factors, such as nutrition, affect them is leading to new opportunities to improve pregnancy rates by influencing follicle development and oocyte quality. This review will focus on follicular development from foetal to adult stages and the influences that nutrition has during some of these developmental stages.

Variation among individual dairy cows in methane measurements made on farm during milking.

The objective of this study was to quantify on-farm variation between and within cows in methane emissions measured during milking, and to determine which factors are related to this variation. Methane emission rate during milking (MERm) was recorded at milking using methane analyzers installed in automatic (robotic) milking stations for 215 cows over a period of 5 mo. Between-cow variation in MERm (mean 2.07, SD 0.629 g/min), was greater than within-cow variation and was related to variation in body weight, milk yield, parity, and week of lactation. Estimation of daily methane emissions from MERm data, using an equation derived from comparisons with respiration chamber data, produced estimates that ranged from 278 to 456 g of CH4/d and were commensurate with values predicted from metabolizable energy requirements for observed body weight and milk yield. It is concluded that methane emissions vary considerably between dairy cows housed under commercial conditions. This variation needs to be taken into account when performing inventories or testing mitigation strategies, but it might offer opportunities for genetic selection.

On-farm methane measurements during milking correlate with total methane production by individual dairy cows.

The objective of this study was to investigate whether measurement of methane emissions by individual dairy cows during milking could provide a useful technique for monitoring on-farm methane emissions. To quantify methane emissions from individual cows on farm, we developed a novel technique based on sampling air released by eructation during milking. Eructation frequency and methane released per eructation were used to estimate methane emission rate. For 82 cows, methane emission rate during milking increased with daily milk yield (r = 0.71), but varied between individuals with the same milk yield and fed the same diet. For 12 cows, methane emission rate recorded during milking on farm showed a linear relationship (R² = 0.79) with daily methane output by the same cows when housed subsequently in respiration chambers. For 42 cows, the methane emission rate during milking was greater on a feeding regimen designed to produce high methane emissions, and the increase compared with a control regimen was similar to that observed for cows in respiration chambers. It was concluded that, with further validation, on-farm monitoring of methane emission rate during milking could provide a low-cost reliable method to estimate daily methane output by individual dairy cows, which could be used to study variation in methane, to identify cows with low emissions, and to test outcomes of mitigation strategies.