Anti-Müllerian hormone levels in serum and testes of male dogs: relations with neuter status and bilateral abdominal cryptorchidism.

Anti-Müllerian hormone (AMH) analysis has contributed to new information in the reproductive endocrinology of domestic animals, due to clinically available diagnostic tools. An accurate and rapid diagnostic method to distinguish between neutered and bilateral abdominal cryptorchid dogs is needed in veterinary practice. Therefore, this study uses an enzyme-linked immunosorbent assay to evaluate the clinical relevance of AMH analysis in peripheral blood as a diagnostic tool for dogs with suspected bilateral abdominal cryptorchidism. The possible alteration of the AMH localization in testicular tissue caused by this pathologic condition was also investigated using immunohistochemistry. Male dogs were divided into three groups of healthy intact (n = 14), healthy castrated (n = 14), and bilateral abdominal cryptorchid (n = 14) dogs. The results demonstrated a higher level of serum AMH in the cryptorchid group compared to the intact group (P < 0.01), while serum AMH levels of all castrated dogs were below the limit of detection (<0.05 ng/mL). Moreover, the percentage of positive AMH immunostaining of the intact group was less than that of the cryptorchid group (P < 0.01). A significantly positive correlation was found between serum AMH concentration and localization in testicular tissues (r = 0.93, P < 0.01). Our findings suggest that AMH levels in the peripheral blood could be used as an alternative and rapid screening method for detecting dogs with abdominal cryptorchidism.

Reproductive aging in male dogs: The epididymal sperm defects and expression of androgen receptor in reproductive tissues.

The present study aimed at investigating age-related changes in epididymal sperm motility, morphology, DNA integrity and androgen receptor (AR) expression in canine reproductive tissues. Fifty-five healthy medium-sized male dogs were divided into four groups: young (1 - 3 years old, n = 14), adult (>3 - 6 years old, n = 12), old (>6 - 9 years old, n = 14) and senile (>9 years old, n = 15). After routine castration, testes, epididymides (head, body and tail) and vas deferens were collected. Spermatozoa were flushed from epididymal tails and their motility, morphology and DNA integrity were examined. Localization of AR was investigated by immunohistochemistry and the positive immunostaining cells were evaluated using image analysis software (NuclearQuant, 3DHISTECH). We found significantly lower percentages of epididymal sperm motility, sperm vigour and viability in adult, old and senile dogs in comparison with young dogs (p < 0.05). Animal's age negatively correlated with epididymal sperm motility, sperm vigour and viability. The primary, secondary, major and minor epididymal sperm defects were significantly higher in senile dogs compared to young dogs. There were positive correlations between age and epididymal sperm defects (p < 0.01). The percentage of sperm with fragmented DNA did not differ between age groups. Testicular AR was expressed in the nucleus of Sertoli cells, Leydig cells and peritubular myoid cells, except for germ cells. Expression of AR was found in all epithelium, lamina propria and smooth muscle cells of the epididymis and vas deferens. Expression levels of AR in testis, epididymis and vas deferens did not differ between age groups (p > 0.05). In conclusion, the present study clearly demonstrated that senescence in dogs was associated with decreased epididymal sperm quality. An age-related increase in the incidence of poor epididymal sperm quality may promote subfertility, especially in senile dogs.

The expressions in oxytocin and sex steroid receptors in the reproductive tissues of normal and unilateral cryptorchid dogs.

In males, oxytocin is involved with various physiological functions, such as reproductive tract contractility and testicular steroidogenesis. Due to the relationship between sex steroid hormones, oxytocin receptor (OTR) expression and cryptorchidism pathogenesis, this study aimed to investigate the mRNA expression and the localization of OTR in relation to sex steroid receptors in the male reproductive tract of both normal and unilateral abdominal cryptorchid dogs using quantitative PCR and immunohistochemistry. Male dogs were divided into two groups of normal and cryptorchid dogs. Samples from each cryptorchid dog were separated into two subgroups: scrotal and abdominal subgroups. The results showed that a lower percentage of positive OTR immunostaining in the testis and epididymis was observed in the cryptorchid group compared to the normal group. Within the cryptorchid group, the mRNA expression and the localization of OTR in the testis and epididymis of the abdominal subgroup was less than that of the scrotal subgroup. Moreover, the localization of OTR and estrogen receptor beta (ERß) in reproductive tissues was positively correlated only in the normal group and not in the cryptorchid group. In conclusion, this study proposed that OTR expression, as well as the correlation between the OTR and ERß in reproductive tissues of male dogs, can be disturbed by cryptorchidism. Furthermore, the OTR, ERß and their correlation may be involved with the pathogenesis of cryptorchidism. Therefore, the study of gene knockout models to confirm the effect of OTR and sex steroid receptors on canine cryptorchidism should be of interest for further investigation.

Expression of oxytocin, progesterone, and estrogen receptors in the reproductive tract of bitches with pyometra.

Canine pyometra is considered a serious and life-threatening condition. Due to the relationship among sex steroid hormones, oxytocin receptor (OTR) expression, and canine pyometra pathogenesis, this study aimed to investigate the expression of oxytocin, progesterone, and estrogen receptors in the reproductive tissues of canines with pyometra by real-time PCR and immunohistochemistry. A total of 27 pyometra bitches were classified into open- and closed-cervix pyometra groups based on the presence of vaginal discharge. Moreover, 15 normal bitches in the luteal phase served as a control group. The results showed that OTR gene expression in the ovary of pyometra bitches was higher than that of normal bitches, whereas the level of OTR gene expression in the cervix of pyometra bitches was less than that of normal bitches (P < 0.05). Conversely, a lower OTR H-score in ovarian follicles was observed in pyometra bitches compared with normal bitches, whereas a higher percentage of OTR-positive immunostaining in uteri and cervices were found in pyometra bitches compared with normal bitches (P < 0.05). Moreover, the H-scores of estrogen receptor alpha in uteri and cervices of pyometra bitches were less than that of normal bitches (P < 0.05). However, the localization of the OTR and sex steroid receptors between groups of pyometra bitches was not different. Our findings suggest that pyometra pathogenesis is associated with a change in expression of OTR and sex steroid receptors in the canine reproductive tract. However, cervical dilation in bitches with pyometra was not influenced by the expression of OTR and sex steroid receptors.

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.