Cyrillus Ververs

Proteome of equine oviducal fluid: effects of ovulation and pregnancy

The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid of the four groups was achieved by isobaric tags for relative and absolute quantification (iTRAQ) labelling and HPLC-tandem mass spectrometry. The presence of an embryo in the ipsilateral oviducal fluid of pregnant mares induced upregulation of 11 and downregulation of two proteins compared with the contralateral side, and upregulation of 19 proteins compared with the ipsilateral side of non-pregnant mares. Several of these upregulated proteins are related to early pregnancy in other species. The present study represents the first high-throughput identification of proteins in the oviducal fluid of the mare. The results support the hypothesis that the equine embryo interacts with the oviduct, affecting the maternal secretion pattern of proteins involved in pregnancy-related pathways.

Malignant Seminoma in Two Unilaterally Cryptorchid Stallions

Two unilateral cryptorchid stallions were referred to the clinic because of chronic debilitating condition with emaciation. Rectal examination, and ultrasound and gross examination revealed in both animals an abdominal mass, caudally of the kidney, and multiple nodules spread over the abdomen. Histologic analysis revealed an intra-abdominal malignant seminoma with intraperitoneal and renal metastasis. Interestingly, a seminoma was also present in the descended testis of the draught horse.

Proteins involved in embryo-maternal interaction around the signalling of maternal recognition of pregnancy in the horse

During maternal recognition of pregnancy (MRP), a conceptus-derived signal leads to the persistence of the corpus luteum and the maintenance of gestation. In the horse, the nature of this signal remains to be elucidated. Several studies have focused on the changes in gene expression during MRP, but little information exists at the protein level. The aim of this study was to identify the proteins at the embryo-maternal interface around signalling of MRP in the horse (day 13) by means of mass spectrometry. A distinct influence of pregnancy was established, with 119 proteins differentially expressed in the uterine fluid of pregnant mares compared to cyclic mares and with upregulation of several inhibitors of the prostaglandin synthesis during pregnancy. By creating an overview of the proteins at the embryo-maternal interface in the horse, this study provides a solid foundation for further targeted studies of proteins potentially involved in embryo-maternal interactions, MRP and pregnancy loss in the horse.

Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration

In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with detached heads. When using intact sperm cells, 8 out of the 25 oocytes cleaved, and 1 developed to the blastocyst stage 9 days after ICSI. None of the oocytes injected with a detached sperm head cleaved. Studies on the paternal influence on ICSI outcome are limited in the horse and further research is needed to define which stallion factors may influence ICSI results. Here, we report the possibility to produce a blastocyst by ICSI of a stallion suffering from testicular degeneration with a poor spermiogram, as long as an intact sperm cell containing a centriole is selected.

Urinary specific gravity as an alternative for the normalisation of endocrine metabolite concentrations in giant panda (Ailuropoda melanoleuca) reproductive monitoring

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal’s changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson’s correlation between creatinine and USpG was high (r = 0.805–0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13–86.93 and 31.61–306.45 ng/mL (USpG correction) versus 2.33–31.20 and 36.36–249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35–3.21 and 0.85–6.80 ng/mL (USpG correction) versus 0.52–10.31 and 2.10–272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

109 REPRODUCTIVE PERFORMANCE PARAMETERS IN A LARGE HERD OF CONFINED FREE-ROAMING WHITE RHINOCEROSES (CERATOTHERIUM SIMUM)

During the last decade, the population of wild white rhinoceroses has been in steady decline, mainly because of increased poaching incidents and habitat loss. Therefore, more data are necessary on reproduction of this endangered species in order to improve captive breeding, which is at present not very successful. Currently, ~20,000 Southern white rhinoceroses are remaining of which the majority are privately owned. The aim of this study was to create reference values of several reproductive parameters for future white rhinoceros breeding. In previous studies, only low numbers of animals have been observed, often in captive settings. In this study performed between 2008 and 2016, reproductive performance was analysed in 1300 animals kept in a geographically identical, confined free-roaming environment. Analyses were performed in R (R Development Core Team, 2008) using the lme4 and fixed package to model the number of animals born (family=Poisson) and sex ratio (family=binomial). Females had a median age of 83.2 months at first calving (interquartile range: 72.9-110.7) and intercalving intervals of 29.2 (interquartile range: 24.6-34.8) months. Fertility records were excellent with 38% adult females calving per year when compared to previous research, in which first reproduction occurred between 78 and 138 months of age with an intercalving period of 3 years average. A clear seasonal calving pattern was seen with a significant increase of calvings during December-April when compared to April-December. In contrast to the Trivers-Willard hypothesis, our results did not show any significant skewed progeny sex ratios. Weather observations showed no significant influence of rain or season on sex ratios of the calves. Furthermore, translocations of animals did not seem to interfere with reproductive success when looking at intercalving periods or age at first calving. In the free roaming environment of over 10,000ha, this captive population showed an average annual population growth (%) of 18±0.07 (minimum 5 to maximum 26). As such, comparable breeding management systems can increase population numbers and contribute to increase dwindling population numbers of the wild white rhinoceros. This is the first study to describe reproductive performances in the white rhinoceros at such large scale, indicating that confined free-roaming populations can be used for captive breeding of white rhinoceros to contribute to white rhinoceros conservation.

Reproductive performance parameters in a large population of game-ranched white rhinoceroses (Ceratotherium simum simum)

The population of free-roaming white rhinoceroses (Ceratotherium simum) is under serious threat. Captive breeding of this species is therefore becoming more important, but this is challenging and often not successful. Obtaining reproductive reference values is a crucial aspect of improving these breeding results. In this study performed between 2008 and 2016, reproductive performance was analysed in 1,354 animals kept in a 8000 hectares game-ranched environment. Descriptive statistics of this captive population showed an average annual herd growth (%) of 7 .0±0.1 (min -9 –max 15). Average calving rates were calculated as an annual calving rate of 20% and biennial calving rate of 37% adult females calving per year. Females had a median age of 83.2 months at first calving (IQR 72.9–110.7) and inter-calving intervals of 29.2 (IQR 24.6–34.8) months. Furthermore, translocations of animals did not interfere with reproductive success in terms of inter-calving periods or age at first calving. Multivariate models showed a clear seasonal calving pattern with a significant increase of the number of calvings during December–April when compared to April–December. Our results did not show any significant skewed progeny sex ratios. Weather observations showed no significant influence of rain or season on sex ratios of the calves.