Induced-ovulation in female dromedary camel involves kisspeptin neuron activation by ß nerve growth factor†.

The dromedary camel (Camelus dromedarius) is a short-day desert breeder in which female ovulation is induced by mating. Current data indicate that male-induced ovulation is triggered by its seminal plasma nerve growth factor beta (ß-NGF), but the exact mechanisms involved in the induction of ovulation are still unknown. In this study, we report that an intramuscular injection of ß-NGF in sexually active short-day-adapted female camels induces an ovulation attested by a surge of circulating LH (2-6 h after treatment) followed by an oocyte release with its cumulus oophorus (confirmed by ultrasonography 72 h after treatment) and a large and progressive increase in circulating progesterone (significant from the 2nd to the 10th days after ß-NGF injection). In addition, this ß-NGF treatment induces a broad nuclear c-FOS activation in cells located in various hypothalamic areas, notably the preoptic area, the arcuate nucleus, the dorso- and ventromedial hypothalamus, the paraventricular nucleus, and the supraoptic nucleus. A double immunostaining with neuropeptides known to be involved in the central control of reproduction indicates that ~28% kisspeptin neurons and 43% GnRH neurons in the proptic area, and ~10% RFRP-3 neurons in the dorso- and ventromedial hypothalamus are activated following ß-NGF injection. In conclusion, our study demonstrates that systemic ß-NGF induces ovulation in the female dromedary camel and indicates that this effect involves the central activation of hypothalamic neurons, notably the kisspeptin neurons.

Clinical and Histological Features of Ovarian Hypoplasia/Dysgenesis in Alpacas.

Alpacas have a high incidence of congenital reproductive tract abnormalities, including ovarian hypoplasia/dysgenesis. Diagnosis of this condition is often challenging. The present study describes the clinical, ultrasonographic, and histologic features of ovarian hypoplasia/dysgenesis syndrome in 5 female alpacas. Additionally, serum AMH levels were compared between female alpacas diagnosed with ovarian hypoplasia/dysgenesis and a group of reproductively sound females (n = 11). The syndrome was suspected based on the presence of an infantile uterus and lack of ovaries by ultrasonography and laparoscopy. All females had normal female karyotype (n = 74 XX), but one presented a minute chromosome. The ovaries from these cases showed 3 main histological classifications: hypoplasia (n = 2), dysgenesis (n = 2), and dysplasia (n = 1). Serum AMH levels in affected females were significantly lower (P < 0.05) than those of reproductively sound control females. In conclusion, Serum AMH level may be helpful in the rapid diagnosis of ovarian hypoplasia/dysgenesis syndrome in alpacas. Furthermore, this syndrome in alpacas presents a variety of histological features. Different mechanisms may be involved in the derangement of ovarian differentiation. Further studies are needed to elucidate the causes of the syndrome.

Molecular Cytogenetic and Y Copy Number Analysis of a Reciprocal ECAY-ECA13 Translocation in a Stallion with Complete Meiotic Arrest.

We present a detailed molecular cytogenetic analysis of a reciprocal translocation between horse (ECA) chromosomes Y and 13 in a Friesian stallion with complete meiotic arrest and azoospermia. We use dual-color fluorescence in situ hybridization with select ECAY and ECA13 markers and show that the translocation breakpoint in ECAY is in the multicopy region and in ECA13, at the centromere. One resulting derivative chromosome, Y;13p, comprises of ECAY heterochromatin (ETSTY7 array), a small single copy and partial Y multicopy region, and ECA13p. Another derivative chromosome 13q;Y comprises of ECA13q and most of the single copy ECAY, the pseudoautosomal region and a small part of the Y multicopy region. A copy number (CN) analysis of select ECAY multicopy genes shows that the Friesian stallion has significantly (p < 0.05) reduced CNs of TSPY, ETSTY1, and ETSTY5, suggesting that the translocation may not be completely balanced, and genetic material is lost. We discuss likely meiotic behavior of abnormal chromosomes and theorize about the possible effect of the aberration on Y regulation and the progression of meiosis. The study adds a unique case to equine clinical cytogenetics and contributes to understanding the role of the Y chromosome in male meiosis.

Recovery of Equine Oocytes in Ambulatory Practice and Potential Complications.

Field collection of oocytes in mares using transvaginal follicular aspiration (TVA) for embryo production has the potential to revolutionate the equine industry. Protocols for TVA in specialized laboratory settings have been described in the scientific literature since the early 1980s. The objective of this study was to determine the success rate of TVA oocytes recovery under ambulatory conditions. A secondary goal of this study was to determine if TVA is associated with any health complications when performed by recently trained practitioners in the field. Follicles (n = 296) from 66 adult clinically healthy mares were aspirated over a period of 6 days. TVAs were performed by 22 veterinarians with 5-20 years of experience in equine and bovine reproductive medicine, but no previous experience in TVA. Oocytes (n = 145) were recovered. No short- or long-term systemic or local complications were observed following TVA in any of the mares used in this study. Fifty-six out of 66 mares became pregnant within 3 months following TVA. This study shows that with proper training, TVA can be successfully used to obtain equine oocytes with no health complications under field conditions in nonspecialized laboratory settings.

An 8.22 Mb Assembly and Annotation of the Alpaca (Vicugna pacos) Y Chromosome.

The unique evolutionary dynamics and complex structure make the Y chromosome the most diverse and least understood region in the mammalian genome, despite its undisputable role in sex determination, development, and male fertility. Here we present the first contig-level annotated draft assembly for the alpaca (Vicugna pacos) Y chromosome based on hybrid assembly of short- and long-read sequence data of flow-sorted Y. The latter was also used for cDNA selection providing Y-enriched testis transcriptome for annotation. The final assembly of 8.22 Mb comprised 4.5 Mb of male specific Y (MSY) and 3.7 Mb of the pseudoautosomal region. In MSY, we annotated 15 X-degenerate genes and two novel transcripts, but no transposed sequences. Two MSY genes, HSFY and RBMY, are multicopy. The pseudoautosomal boundary is located between SHROOM2 and HSFY. Comparative analysis shows that the small and cytogenetically distinct alpaca Y shares most of MSY sequences with the larger dromedary and Bactrian camel Y chromosomes. Most of alpaca X-degenerate genes are also shared with other mammalian MSYs, though WWC3Y is Y-specific only in alpaca/camels and the horse. The partial alpaca Y assembly is a starting point for further expansion and will have applications in the study of camelid populations and male biology.

Effects of Feeding Coenzyme Q10-Ubiquinol on Plasma Coenzyme Q10 Concentrations and Semen Quality in Stallions.

Although coenzyme Q10 (CoQ10) serves as an antioxidant and energy source for spermatozoa when added to stallion semen before cooling or freezing, the effects of feeding CoQ10 on semen quality have not been studied. We assessed the effects of daily oral ingestion of CoQ10-ubiquinol by stallions on their plasma CoQ10 concentrations and semen quality. Seven mature Andalusian stallions ate 1g ubiquinol/day for 4 weeks followed by a 4-week washout period. Four horses initially completed an additional 4-week control period without ubiquinol. Blood was sampled weekly for determination of plasma CoQ10 concentrations. Ejaculates were collected every two weeks and assessed for total motility (TM), progressive motility (PM), and viability (V) after cooling for 24hours (T1), immediate cryopreservation (T2), and cryopreservation after 24hours cooling (T3). Ingesting ubiquinol resulted in an increase in plasma CoQ10 concentration (P < .001). Two weeks of CoQ10-ubiquinol resulted in improved V with all treatments (T1: P = .007; T2: P = .05; T3: P = .01) and PM with T3 (P = .04). In five stallions, TM and PM were also improved for T1 (P = .01 and P = .02, respectively) and TM increased with T2 (P = .03). Overall, semen quality parameters increased within the first 2 weeks of supplementation, plateaued at the end of the 4-week supplementation period and persisted after discontinuing ubiquinol until the end of the sampling period (8 weeks). Feeding 1 g CoQ10-ubiquinol for 4 weeks to breeding stallions improved semen quality after cooling and freezing in 5 of 7 stallions. This could be important for improving reproductive efficiency in stallions.

Investigation of male and female infertility in llamas and alpacas.

Llamas and alpacas are important production animals in South America, with increasing interest in other parts of the world. Poor reproductive efficiency combined with several unique anatomical and physiological reproductive features offer challenges in the diagnosis and treatment of infertility in camelids. This review presents an approach to the clinical investigation and common causes of infertility and subfertility in the male and female. The selection of males for breeding should be made based on complete evaluation to eliminate congenital and possibly hereditary disorders. Common disorders of the male reproductive system include testicular hypoplasia, testicular and epididymal cysts and testicular degeneration. Semen evaluation presents some challenges owing to the viscous nature of the ejaculate in these species. Females should be screened for congenital genital defects before breeding. Causes of subfertility in the female are dominated by ovarian and uterine disorders. A systematic clinical approach and the use of endometrial biopsy and advanced techniques, such as laparoscopy, allow early identification of these disorders. Further research is needed for continued understanding of the reproductive pathological processes in these species.

Basal Levels and hCG Responses of Serum Testosterone and Estrogen in Male Alpacas.

Steroid response to human Chorionic Gonadotropin (hCG) administration has been used in various species to study testicular function and for diagnostic purposes. In this study, two experiments were conducted to determine serum testosterone concentration response to administration of hCG and its correlation with testicular weight. In the second experiment the relationship between age, testosterone and estrogen response to hCG, and testicular histometry was in pre-pubertal and post-pubertal male alpacas. For experiment 1, males in two age groups (2 to 3 years, n = 9) and (4 to 7 years; n = 15) received 3,000 IU hCG IV, 36 to 48 h before castration. Serum testosterone concentration was determined before (T0), 1 h (T1), 2 h (T2), 8 h (T8), and 24 h (T24) after administration of hCG. Basal concentrations of serum testosterone was significantly different (P < 0.01) between age groups. Serum testosterone concentrations increased over time and doubled 2 h after treatment. The highest change (250 to 300% increase from T0) was observed at 8 h (3.5 ± 0.3 ng/ml). A significant correlation (P < 0.01; r = 0.64) was found between serum testosterone concentration and total testicular weight. For experiment 2, 60 males ranging in age from 6 to 60 months were used. Serum testosterone and estrogen was determined in samples taken just prior to and 2 h after administration of 3,000 IU hCG. Basal serum testosterone concentrations were very low (≤0.1 ng/mL) until 9 months of age then increased steadily with age. There was a significant variation amongst males within the same age group. Serum testosterone concentration increased by 2- to 4-fold 2 h after hCG injection (P ≤ 0.05). Males in the 13 to 14 months of age group had the highest rise. Estrogen concentration increased in response to hCG administration and was detected only in males with high testosterone. We conclude that administration of 3,000 IU of hCG IV can be used reliably to detect testicular tissue and study its steroidogenic activity. The response is correlated with testicular weight and Leydig cell number. Testicular growth and sensitivity to LH stimulation increases between the ages of 13 and 14 months. The aromatizing ability of Leydig cells increased significantly in post-pubertal male alpacas.

Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Dromedary camel: A model of heat resistant livestock animal.

Camels are highly adapted to harsh environments. The dromedary camel is adapted to a wide range of arid and semi-arid conditions. The aim of the present paper is to review some of the key adaptation characteristics of the dromedary and how they affect reproductive patterns. Special attention is given to the reproductive seasonality and interaction between lactation and reproduction. Adaptive mechanisms are described including some of the recent molecular aspects with respect to heat shock protein expression in camels.

Azoospermia and Y Chromosome-Autosome Translocation in a Friesian Stallion.

This case report describes spermatogenic arrest and azoospermia in a stallion with a unique Y chromosome-autosome translocation. Clinical diagnosis of azoospermia was based on history of infertility and evaluation of ejaculates collected for artificial insemination. Clinical and ultrasonographic evaluation of the external and internal genitalia did not reveal any abnormalities except for smaller than normal testicular size. Azoospermia of testicular origin was confirmed by determining alkaline phosphatase concentration in semen. Histological evaluation of testicular tissue after castration confirmed early spermatogenic arrest. Cytogenetic evaluation showed the presence of translocation between the Y chromosome and chromosome 13. To the authors' knowledge, this is the first case of azoospermia with a cytogenetically detected Y chromosome abnormality, suggesting that the horse Y chromosome may carry sequences critical for normal spermatogenesis.

Chromosome-Level Alpaca Reference Genome VicPac3.1 Improves Genomic Insight Into the Biology of New World Camelids.

The development of high-quality chromosomally assigned reference genomes constitutes a key feature for understanding genome architecture of a species and is critical for the discovery of the genetic blueprints of traits of biological significance. South American camelids serve people in extreme environments and are important fiber and companion animals worldwide. Despite this, the alpaca reference genome lags far behind those available for other domestic species. Here we produced a chromosome-level improved reference assembly for the alpaca genome using the DNA of the same female Huacaya alpaca as in previous assemblies. We generated 190X Illumina short-read, 8X Pacific Biosciences long-read and 60X Dovetail Chicago® chromatin interaction scaffolding data for the assembly, used testis and skin RNAseq data for annotation, and cytogenetic map data for chromosomal assignments. The new assembly VicPac3.1 contains 90% of the alpaca genome in just 103 scaffolds and 76% of all scaffolds are mapped to the 36 pairs of the alpaca autosomes and the X chromosome. Preliminary annotation of the assembly predicted 22,462 coding genes and 29,337 isoforms. Comparative analysis of selected regions of the alpaca genome, such as the major histocompatibility complex (MHC), the region involved in the Minute Chromosome Syndrome (MCS) and candidate genes for high-altitude adaptations, reveal unique features of the alpaca genome. The alpaca reference genome VicPac3.1 presents a significant improvement in completeness, contiguity and accuracy over VicPac2 and is an important tool for the advancement of genomics research in all New World camelids.

Enhanced sperm production in bulls following transient induction of hypothyroidism during prepubertal development.

Male reproductive capacity is a critical component of cattle production and the majority of genetic gain is made via selective utilization of gametes from desirable sires. Thus, strategies that enhance sperm production increase the availability of elite genetics for use in improving production characteristics of populations on a worldwide scale. In all mammals, the amount of sperm produced is strongly correlated to the number of Sertoli cells in testes. Studies with rodents showed that the size of the Sertoli cell population is set during prepubertal development via signaling from thyroid hormones. Here, we devised a strategy to increase Sertoli cell number in bulls via induction of a transient hypothyroidic state just prior to and extending beyond the period of Sertoli cell proliferation that we found to normally cease between 4.5 and 5 mo of age. In adulthood, these bulls produced a significantly greater number of sperm compared to age-matched controls and their testes contained nearly 2 times more Sertoli cells. Importantly, sperm motility, morphology, fertilizing ability, and viability after cryopreservation were found to be no different for treated bulls compared to untreated control bulls. This strategy of transient induction of hypothyroidism during a defined period of prepubertal development in bulls could prove to be an efficacious approach for enhancing daily sperm production in genetically desirable sires that will, in turn, provide an avenue for improving the efficiency of commercial cattle production.

Effect of Melatonin Implants during the Non-Breeding Season on the Onset of Ovarian Activity and the Plasma Prolactin in Dromedary Camel.

To examine a possible control of reproductive seasonality by melatonin, continual-release subcutaneous melatonin implants were inserted 4.5 months before the natural breeding season (October-April) into female camels (Melatonin-treated group). The animals were exposed to an artificial long photoperiod (16L:8D) for 41 days prior to implant placement to facilitate receptivity to the short-day signal that is expected with melatonin implants. The treated and control groups (untreated females) were maintained separately under outdoor natural conditions. Ovarian follicular development was monitored in both groups by transrectal ultrasonography and by plasma estradiol-17ß concentrations performed weekly for 8 weeks and then for 14 weeks following implant insertion. Plasma prolactin concentrations were determined at 45 and 15 days before and 0, 14, 28, 56, and 98 days after implant insertion. Plasma melatonin concentration was determined to validate response to the artificial long photoperiod and to verify the pattern of release from the implants. Results showed that the artificial long photoperiod induced a melatonin secretion peak of significantly (P < 0.05) shorter duration (about 2.5 h). Melatonin release from the implants resulted in higher circulating plasma melatonin levels during daytime and nighttime which persisted for more than 12 weeks following implants insertion. Treatment with melatonin implants advanced the onset of follicular growth activity by 3.5 months compared to untreated animals. Plasma estradiol-17ß increased gradually from the second week after the beginning of treatment to reach significantly (P < 0.01) higher concentrations (39.2 ± 6.2 to 46.4 ± 4.5 pg/ml) between the third and the fifth week post insertion of melatonin implants. Treatment with melatonin implants also induced a moderate, but significant (P < 0.05) suppressive effect on plasma prolactin concentration on the 28th day. These results demonstrate that photoperiod appears to be involved in dromedary reproductive seasonality. Melatonin implants may be a useful tool to manipulate seasonality and to improve reproductive performance in this species. Administration of subcutaneous melatonin implants during the transition period to the breeding season following an artificial signal of long photoperiod have the potential to advance the breeding season in camels by about 2.5 months.

Effect of age and castration on serum anti-Müllerian hormone concentration in male alpacas.

The synthesis of anti-Müllerian Hormone (AMH) by the Sertoli cells in males is crucial for sexual differentiation. There are no studies on AMH in Camelids. The objectives of this research were to 1) compare AMH serum concentrations in prepubertal and adult male alpacas and 2) determine the effect of castration on these concentrations in adult males to provide a validation of a commercial AMH test in alpacas. Serum samples were obtained from 15 prepubertal animals (5 for each age groups of 6, 7 and 8 months) and from 5 adult males (age 5-9 years), pre- and 24 h post-castration. AMH was determined with a quantitative ELISA according to the manufacture's instructions. There was not significant difference (P < 0.05) in AMH level (pg/ml) between pre-pubertal (549.9 ± 120.8, 789.4 ± 172.3, 597.5 ± 177.3 for ages 4, 7, and 8 months, respectively) and adult alpacas (938.7 ± 175.9). In adult males, AMH concentration decreased significantly following castration (P < 0.05) (938.7 ± 383.5 pg/ml) pre-castration, and 222.1 ± 116.5 pg/ml) after castration). There was a positive correlation between testosterone levels and AMH. In conclusion, the quantitative assay used is a reliable test to determine AMH in alpacas. The AMH level in prepubertal and adult alpacas appear to not differ, contrarily from other mammals, this requires further investigation. The decrease in serum AMH concentrations after castration suggests that measurement of this hormone can be used to diagnose bilateral cryptorchid or hemicastrated unilateral cryptorchid animals in this species.

Monitoring and controlling follicular activity in camelids.

This paper reviews that state of our knowledge concerning follicular wave dynamics, monitoring and manipulation. All camelids have overlapping follicular waves in absence of ovulation which is induced by a seminal plasma factor (ßNGF). The interval between follicular waves varies. The size of the ovulatory follicle varies between 11 and 25 mm in camels and between in 6 and 13 mm in South American Camelids. The interval between induction of ovulation and next ovulatory follicle is 15 ± 1 day for all camelids. Follicular activity is best monitored by transrectal ultrasonography. Progesterone therapy for 7-15 days seems to suppress follicular dominance but does not completely inhibit follicular recruitment. Combination of estradiol and progesterone seems to provide better control of follicular activity. Both methods have provided variable results in the synchronization of follicular waves. Combination of induction of ovulation with GnRH and luteolysis at predetermined times shows some promise in synchronization of follicular dominance. These synchronization protocols require further investigation in order to provide practical approaches for fixed-time breeding. Ovarian superstimulation with FSH and eCG alone or in combination is somewhat successful. The best results are obtained when treatment is initiated at the emergence of a new follicular wave after induction of ovulation or following treatment with progesterone for 7-14 days. However, response remains extremely variable particularly in terms of ovulation rate and number of recovered embryos. Sources of this variability need to be studied including the effects of season, nutrition, doses and frequency of administration of gonadotropin.

Oxytocin is not involved in luteolysis and early maternal recognition of pregnancy (MRP) in alpacas.

Pregnancy maintenance depends on the maternal recognition of pregnancy (MRP), a physiological process by which the lifespan of the corpus luteum is prolonged. This mechanism is not well characterized in camelids. The objectives of the present research were to determine if exogenous oxytocin prolongs the corpus luteum activity in alpacas and to evaluate expression and localization of oxytocin receptors within the endometrium at 9 and 14days post-mating. In the oxytocin studies, plasma progesterone profiles were determined after ovulation in the same alpacas on 2 cycles: one cycle without oxytocin treatment and one cycle with oxytocin treatment. Oxytocin was administered daily by intramuscular injections (IM) at a dose of 20IU (experiment 1, n=6) or 60IU (experiment 2, n=7 from day 3 through day 10 after induction of ovulation with GnRH IM. There was no significant difference in the length of the luteal phase (i.e. corpus luteum lifespan) between the treated and control cycles using either 20 or 60IU of oxytocin. In the final experiment, uteri from open and pregnant alpacas (n=4 per group) at 9 and 14days post-mating were evaluated for expressions of oxytocin receptors by immunohistochemistry. No significant difference (P≤0.05) in the expression of oxytocin receptors was observed between open and pregnant animals in either staining intensity or tissue localization. We conclude that oxytocin is not involved in luteolysis and early MRP in alpacas.

Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene.

Genome editing tools have revolutionized the generation of genetically modified animals including livestock. In particular, the domestic pig is a proven model of human physiology and an agriculturally important species. In this study, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.

Precise gene editing paves the way for derivation of Mannheimia haemolytica leukotoxin-resistant cattle.

Signal peptides of membrane proteins are cleaved by signal peptidase once the nascent proteins reach the endoplasmic reticulum. Previously, we reported that, contrary to the paradigm, the signal peptide of ruminant CD18, the ß subunit of ß2 integrins, is not cleaved and hence remains intact on mature CD18 molecules expressed on the surface of ruminant leukocytes. Leukotoxin secreted by Mannheimia (Pasteurella) haemolytica binds to the intact signal peptide and causes cytolysis of ruminant leukocytes, resulting in acute inflammation and lung tissue damage. We also demonstrated that site-directed mutagenesis leading to substitution of cleavage-inhibiting glutamine (Q), at amino acid position 5 upstream of the signal peptide cleavage site, with cleavage-inducing glycine (G) results in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of target cells. In this proof-of-principle study, we used precise gene editing to induce Q(‒5)G substitution in both alleles of CD18 in bovine fetal fibroblast cells. The gene-edited fibroblasts were used for somatic nuclear transfer and cloning to produce a bovine fetus homozygous for the Q(‒5)G substitution. The leukocyte population of this engineered ruminant expressed CD18 without the signal peptide. More importantly, these leukocytes were absolutely resistant to leukotoxin-induced cytolysis. This report demonstrates the feasibility of developing lines of cattle genetically resistant to M. haemolytica-caused pneumonia, which inflicts an economic loss of over $1 billion to the US cattle industry alone.