Effects of Gonadotrophin Releasing Hormone Administration on the Pituitary-Ovarian Axis in Anoestrous vs Ovariectomized Bitches

The aim of this study was to determine the effects of gonadotrophin releasing hormone (GnRH) administration on the plasma concentrations of reproductive hormones in intact and ovariectomized (OVX) bitches. Therefore, blood samples were collected at multiple times before and after the administration of 10 microg/kg GnRH (Fertagyl)) for the determination of the plasma concentrations of luteinizing hormone (LH), oestradiol, progesterone and testosterone in six anoestrus and in six OVX bitches. The mean plasma LH concentrations before and 60 min after GnRH administration were significantly lower in the anoestrous bitches than in the OVX bitches. In both groups GnRH administration resulted in a significant increase in the plasma LH concentration. The highest plasma LH concentrations were found at 10 min after GnRH administration and these values did not differ significantly between the two groups. Only in the anoestrous bitches a significant increase in plasma oestradiol concentrations was found after GnRH administration and these values were significantly higher than those in the OVX bitches. The plasma concentrations of progesterone and testosterone were low (close to or below the limit of quantitation) both before and after GnRH administration and the differences between anoestrous and OVX bitches were not significant. It can be concluded that (i) basal plasma LH concentration is significantly higher in OVX bitches than in anoestrous bitches, (ii) plasma LH concentration increases after GnRH administration in both anoestrous and OVX bitches, (iii) GnRH administration causes a significant rise in plasma oestradiol concentration only if ovarian tissue is present and (iv) measurement of plasma progesterone and testosterone concentrations before and after GnRH administration does not aid in distinguishing between anoestrous and OVX bitches. The results of this study may provide a basis for the diagnosis of remnant ovarian tissue and verification of neuter status in the bitch.     

Methods for Equine Preantral Follicles Isolation: Quantitative Aspects

The aim of this study was to test the use of mechanical and mechanical‐enzymatic methods, saline solution (SS), and PBS solution for the manipulation and isolation of mare ovarian preantral follicles (PAFs). The ovaries were subjected to mechanical isolation (mixer) alone or in association with enzymatic digestion (collagenase). Incubation times of 10 and 20 min were employed. In the first group, 4.1 ± 4.9 PAFs were harvested with the mechanical‐enzymatic method vs 71.1 ± 19.2 with the mechanical procedure, showing a significant difference between methods; using SS and PBS, these numbers were 35.7 ± 34.3 and 39.6 ± 39.6, respectively, with no significant difference between solutions. In the second group, there was significant difference between methods, with 7.1 ± 10.6 follicles harvested with the mechanical‐enzymatic method vs 63.2 ± 22.9 with the mechanical procedure; using SS and PBS, means were 35.5 ± 36.4 and 34.9 ± 31.1, respectively. The mechanical method proved more effective than the mechanical‐enzymatic approach. Both SS and PBS can be used as a media for equine PAFs preparation.     

Anoestrus in the Dog: a Fascinating Story

In the bitch, progression from early to late anoestrus is characterized by a higher amplitude and a larger number of gonadotrophin-releasing hormone (GnRH) pulses generated by the hypothalamus, an increase in the sensitivity of the pituitary to GnRH, and an increase in ovarian responsiveness to gonadotrophins. An increase in basal plasma follicle-stimulating hormone (FSH) concentration is a critical event required for initiation of folliculogenesis. A period of increased luteinizing hormone pulsatility has been reported shortly before the onset of pro-oestrus. Apart from these changes in the hypothalamic-pituitary-ovarian axis, there is also involvement of dopaminergic influences in the initiation of a new follicular phase in the bitch. Administration of the dopamine-agonists bromocriptine and cabergoline shortens the anoestrus and is associated with a decrease in the plasma prolactin concentration, suggesting that the shortened anoestrus is the result of suppression of prolactin secretion. Yet, the anoestrus is not shortened in bitches treated with metergoline, a drug, which in a low dosage lowers the plasma prolactin concentration via a serotonin-antagonistic pathway. In addition, under physiological conditions low plasma prolactin concentrations are found during the entire anoestrus. Furthermore, a low dosage of bromocriptine, insufficient to cause a decrease in the plasma prolactin concentration, prematurely induces folliculogenesis. These observations indicate that not the decrease in the plasma prolactin concentration, but another dopamine-agonistic influence plays a critical role in the transition to a new follicular phase. The dopamine-agonist induced shortening of the anoestrus is associated with a rapid rise in the basal plasma FSH concentration, similar to what is observed during the physiological late anoestrus. This observation underlines the importance of an increase in the circulating plasma FSH concentration in the initiation of folliculogenesis in the bitch.     

Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium‐regulating multifunctional protein

Regucalcin (RGN) is a calcium‐regulating, anti‐apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real‐time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34‐kDa, 28‐kDa and 24‐kDa isoforms, wherein the 34‐kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium‐related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.     

Research progress on the role of melatonin and its receptors in animal reproduction: A comprehensive review

Melatonin and its receptors play a crucial role in the regulation of the animal reproductive process, primarily in follicular development. However, the role that melatonin performs in regulating hormones related with reproduction remains unclear. Melatonin and its receptors are present both in female and male animals’ organs, such as ovaries, heart, brain and liver. Melatonin regulates ovarian actions and is a key mediator of reproductive actions. Melatonin has numerous effects on animal reproduction, such as protection of gametes and embryos, response to clock genes, immune‐neuroendocrine, reconciliation of seasonal variations in immune function, and silence or blockage of genes. The growth ratio of reproductive illnesses in animals has raised a remarkable concern for the government, animal caretakers and farm managers. In order to resolve this challenging issue, it is very necessary to conduct state‐of‐the‐art research on melatonin and its receptors because melatonin has considerable physiognomies. This review article presents a current contemporary research conducted by numerous researchers from the entire world on the role of melatonin and its receptors in animal reproduction, from the year 1985 to the year 2017. Furthermore, this review shows scientific research challenges related to melatonin receptors and their explanations based on the findings of 172 numerous research articles, and also represents significant proficiencies of melatonin in order to show enthusiastic study direction for animal reproduction researchers.     

Co‐culture of Buffalo Preantral Follicles with Different Somatic Cells

The effect of co‐culture of buffalo preantral follicles (PFs) with different somatic cells, i.e, cumulus, granulosa, ovarian mesenchymal and oviductal epithelial cells was studied. Large PFs (250–450 μm) were isolated by microdissecting the trypsin (1%) digested ovarian cortical slices. Cumulus cells were isolated by repeated pipetting of oocytes, granulosa cells were isolated by aspirating from punctured PFs and ovarian mesenchymal cells were isolated from ovarian cortex by scraping the cortical slices and passing through 20 μm filter. Preantral follicles were cultured in standard culture medium without somatic cells or co‐cultured with cumulus cells, granulosa cells, ovarian mesenchymal cells and oviductal epithelial cells for 80 days. The growth rate (μm/day) of the PFs was monitored by measuring follicular diameter on day 0, 30, 60 and 80 days of culture. The viability of PFs was evaluated by trypan blue staining. The results indicated that PFs co‐cultured with cumulus, granulosa and ovarian mesenchymal cells had a better development and survivality compared with control and those co‐culture with oviductal epithelial cells. Maximum growth and survivality of PFs were achieved when cultured with cumulus cells. It is concluded that inclusion of somatic cells in PF culture media had beneficial effect on the growth of PFs and cumulus cells supported maximum growth and survivality of PFs in vitro of all somatic cells tested.     

Lactoferrin increases sperm membrane functionality of frozen equine semen

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome‐reacted sperm were evaluated with a computer‐assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.     

Apparent lack of efficacy of toltrazuril against Eimeria species affecting brown kiwi (Apteryx mantelli) at a captive rearing facility

AIM: To assess the efficacy of toltrazuril against the Eimeria spp. affecting brown kiwi (Apteryx mantelli).

METHODS: Droppings were collected from three brown kiwi, aged <6 months old, at a captive rearing facility in the North Island of New Zealand, between 22 February and 20 April 2017, on 14 sampling dates. Only droppings (n=30) that were excreted between 03:00 and 07:00, as determined using video surveillance, were included for analysis, reflecting the peak time for shedding of coccidial oocysts for brown kiwi. Oocysts were quantified in each sample and Eimeria species identified on the basis of oocyst morphology. All samples were collected between 2 and 10 days after the birds had been treated with 25?mg/kg toltrazuril.

RESULTS: Eimeria spp. oocysts were identified in 28/30 individual samples and on 14/14 sampling dates. Oocyst counts varied from 0 to 328,080 oocysts per gram (opg), and at least one oocyst count >10,000 opg was measured on 12/14 sampling dates. Three species of Eimeria were observed, with Eimeria apteryxii and E. kiwii most commonly encountered, whereas only one sample contained E. paraurii.

CONCLUSIONS AND CLINICAL RELEVANCE: In the three birds monitored at this research site, there was a high abundance of E. apteryxii and E. kiwii oocysts in droppings despite recent administration of toltrazuril. These results suggest that the populations of Eimeria spp. affecting brown kiwi at this location appear to possess an ability to survive exposure to toltrazuril. Toltrazuril is widely used at captive rearing facilities to limit the effects of coccidiosis in juvenile kiwi. If a lack of efficacy is confirmed, it will be necessary to investigate alternative treatment regimens alongside broader environmental management strategies.