Sayamon Srisuwatanasagul

Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity.

The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P < 0.05). Morphologically normal primordial follicles were higher in the sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P < 0.05). Moreover, less apoptotic primordial follicles were found in both sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P < 0.05). However, their Cx 43 expression showed no difference among the groups of different sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P < 0.05). In addition, the slow freezing resulted in more postthawed morphologically normal primordial follicles (38.8% vs. 28.3%, P < 0.05) and less apoptotic primordial follicles (3.8% vs. 8.9%, P < 0.05) than vitrification. The Cx 43 expression showed no difference between slow freezing and vitrification. The present study reported the positive effects of sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues.

Distribution of estrogen receptor alpha and progesterone receptor, and leukocyte infiltration in the cervix of cyclic bitches and those with pyometra

The objectives were to localize estrogen receptor alpha (ERα) and progesterone receptor (PR), and enumerate leukocyte infiltration in cervical tissue of normal bitches during various stages of the estrous cycle (n = 35), as well as in those developing open (n = 22) or closed-cervix pyometra (n = 19). Each pyometra group was subdivided into anestrus and diestrus. Cervical tissues were collected after ovariohysterectomy. Receptor expressions were determined by immunohistochemistry and leukocyte infiltration was evaluated in histological sections stained with haematoxylin-eosin. The assessment was performed in two parts of cervical sections: the uterine part in four tissue layers (surface epithelium (SE), lamina propria (LP), glandular epithelium (GE), and tunica muscularis (M)), and the vaginal part in three layers (SE, LP and M). An immunohistochemical total score consisted of the addition of both the intensity and proportional scores. The ERα and PR scores differed between groups (P < 0.05) and between layers (P < 0.05), but were not significantly different between uterine and vaginal parts. The ERα score was lowest in the open-cervix pyometra bitches at anestrus and in closed-cervix pyometra bitches at diestrus. For all types of immune cells, there were no significant differences among stages of the estrous cycle in normal bitches, whereas neutrophils were lower in both sub-groups of closed-cervix versus open-cervix pyometra (P < 0.05). In conclusion, distributions of ERα and PR were similar along the longitudinal axis of the canine cervix. We inferred that cervical dilation in normal bitches and bitches with uterine pathology was likely controlled by different mechanisms. Receptor expressions were influenced by stage of the estrous cycle in normal bitches, whereas neutrophil infiltration in cervical tissue appeared to be involved in cervical dilation in bitches with pyometra, regardless of estrous stages.

Sperm quality and the morphology of cryopreserved testicular tissues recovered post-mortem from diverse wild species.

This study compared the effects of slow and fast freezing of testicular tissue of wild animals collected at post-mortem on testicular structure and testicular sperm. The testes of seven animals that had died in captivity; three felids (jungle cat, lion and leopard), two cervids (rusa deer and feas muntjac) and two bovids (Sumatran serows) were cryopreserved using slow- and fast-freezing protocols. There were greater reductions in the integrity of the sperm membrane and DNA in tissues cryopreserved using slow freezing compared to fast freezing (membrane integrity reduced by 21.5 ± 12.4% vs. 13.0 ± 6.9%, P = 0.11 and DNA integrity reduced by 22.7 ± 16.3% vs. 6.6 ± 6.3%, P = 0.13). Histologically, there were similar degrees of detachment and shrinkage of the seminiferous tubules whereas, TUNEL assay revealed a tendency towards more apoptotic changes in the intra-tubular cells of tissues frozen using fast freezing compared to slow freezing (P = 0.09). In conclusion, fast freezing tended to cause less damage to testicular sperm but its protective effect on intra-tubular cells was likely compromised. This is the first report of gamete recovery in the wild and of the comparison in various wildlife species, between testicular tissues cryopreserved using different protocols.

Collagen and Glycosaminoglycan Profiles in the Canine Cervix during Different Stages of the Estrous Cycle and in Open- and Closed-Cervix Pyometra

ABSTRACT The extracellular matrix of the cervix that comprises collagen, elastin, proteoglycans and glycosaminoglycans (GAGs) is thought to have an essential role in cervical relaxation. This study investigated the proportion of collagen and smooth muscle as well as the GAGs in cervices obtained from healthy bitches at different stages of the estrous cycle and bitches with open- and closed-cervix pyometra. Cervices were collected after ovariohysterectomy. The proportion of collagen to smooth muscle was determined using Masson’s trichrome staining. Alcian blue staining was used to evaluate the relative distribution of cervical GAGs. The proportion of cervical collagen relative to smooth muscle was higher at estrus compared to anestrus (P≤0.05). It was also higher (P≤0.05) in bitches with open- compared to those with closed-cervix pyometra. Overall, hyaluronan (HA) was the predominant GAG in the canine cervix. In the luminal epithelium, the staining intensity for HA was stronger in estrus than in anestrus (P≤0.05), but not in diestrus (P>0.05). On the contrary, the intensity for the combined keratan sulfate (KS) and heparan sulfate (HS) was stronger in anestrus than in estrus and diestrus (P≤0.05). In bitches with pyometra, the staining intensity of the stroma for KS and HS was weaker in open- compared to closed-cervix pyometra (P≤0.05). Collectively, the different profiles of collagen and GAG suggest that the metabolism of both collagen and GAGs in the canine cervix is associated with hormonal statuses during the estrous cycle and cervical patency of bitches with pathological uterine conditions, such as pyometra.

Expression of Progesterone Receptor in the Utero‐tubal Junction After Intra‐uterine and Deep Intra‐uterine Insemination in Sows

The aim of this study was to investigate the expression of progesterone receptor (PR) in the utero-tubal junction (UTJ) of sows at 24 h after intra-uterine insemination (IUI) and deep intra-uterine insemination (DIUI) compared with conventional artificial insemination (AI) in pigs. Fifteen multiparous sows were used: AI (n = 5), IUI (n = 5) and DIUI (n = 5). The sows were inseminated with a single dose of diluted semen during the second oestrus after weaning at 6-8 h prior to ovulation (AI: 3000 × 10(6) spermatozoa, IUI: 1000 × 10(6) spermatozoa and DIUI: 150 × 10(6) spermatozoa). The UTJ was collected and subject to immunohistochemical staining using avidin-biotin immunoperoxidase technique with mouse monoclonal antibody to PR. In the oviductal part of the UTJ, the intensity of PR in the tunica muscularis and the proportion of PR-positive cells in the surface epithelium after DIUI were lower than AI (p < 0.05). The intensity and the proportion of PR-positive cells between AI and IUI in all compartments of the UTJ did not differ significantly (p > 0.05). When comparing between tissue compartments, prominent staining was observed in the muscular layer of the UTJ. It could be concluded that the expression of PR in the UTJ prior to fertilization after DIUI with a reduced number of spermatozoa was lower than that after AI. This might influence sperm transportation and the fertilization process.

The number of ERα and PR in the mammary glands of bitches with and without tumor mass using immunohistochemical assay

The objectives of this study were to evaluate the expressions of estrogen receptors alpha (ERα) and progesterone receptors (PR) in histologically normal and tumoral canine mammary tissues within the same bitch and to correlate between the immunohistochemical staining scores of both receptors and duration of tumor growth. Twelve tumoral and contralateral normal mammary tissues, both determined by histology were surgically obtained from 12 dogs. The expressions of ERα and PR were evaluated by avidin–biotin–peroxidase complex (ABC) method and ERα and PR scores were calculated. The expressions of the ERα and the PR between normal and tumoral mammary tissues were not significantly different. The PR expression and duration of tumor growth showed a significantly inverse correlation. A positive correlation was found between the number of the ERα and the PR in both normal and tumoral mammary tissues which may indicate that either ERα or PR could be used as a prediction of hormonal therapy parameter in dogs. However, the information of ERα and PR status as well as hormonal therapy is not routinely available in veterinary medicine and it appears that half of the mammary tumors from the present study are hormone-independent tumors (ERα and PR negative). Therefore, the treatment of canine mammary tumor by hormonal therapy should be carefully considered.

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 (ERβ) 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 ERβ in reproductive tissues of male dogs, can be disturbed by cryptorchidism. Furthermore, the OTR, ERβ 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.

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary.

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.

GnRH-agonist implantation of prepubertal male cats affects their reproductive performance and testicular LH receptor and FSH receptor expression.

This study was conducted to investigate the effect of GnRH-agonist implantation in prepubertal tomcats on sexual behavior, reproductive performance, and expression of testicular LH receptor (LHR) and FSH receptor (FSHR) and also to compare the testicular characteristics, LHR and FSHR expression between prepubertal and adult tomcats. In experiment 1, 3-month-old tomcats (n = 6/group) were either treated with or left without 4.7 mg deslorelin implants. Semen collection and evaluation were performed just before castration at 48 weeks after treatment; removed testes were analyzed for mRNA and protein expression of LHR and FSHR. We were able to collect semen from six non-treated cats, whereas in treated cats, semen was uncollectable. The results revealed that sexual behavior was absent in the implanted cats throughout the study period. Testicular volume was found to decrease from 30 weeks after treatment onward in the implanted cats compared to the controls (P < 0.05). Semen production was found only in non-implanted cats. Testicular tissue score, seminiferous tubule diameter, and LHR protein expression were found lower in the implanted cats (P < 0.05), but no differences were observed in mRNA expression of LHR and protein expression of FSHR between groups. The mRNA expression of FSHR was higher in the implanted (P < 0.05) compared to control cats. In experiment 2, testes from prepubertal (n = 6) and adult (n = 6) male cats were collected after castration and analyzed for mRNA and protein expression of LHR and FSHR. No differences were observed in the protein expression of LHR and FSHR between the two groups, whereas mRNA expression of FSHR was higher in prepubertal cats (P < 0.05). Testicular and epididymal weight, diameter of seminiferous tubules, and the testicular grade were higher in the adult compared to prepubertal cats (P < 0.05). In conclusion, deslorelin implants suppressed protein expression of LHR and enhanced mRNA expression of FSHR along with suppression of reproductive function without any adverse effects for at least 48 weeks in male cats.

Proliferation and apoptosis in normal bitch mammary tissues in relation to progesterone level

The objective of this study was to investigate the proliferating activity and apoptosis in canine normal mammary tissues in different cycle stages. Mammary tissues were collected from five beagle bitches at six different estrous stages: anestrus, proestrus, estrus, early diestrus, mid diestrus, and late diestrus. The expression of Ki-67 protein and apoptotic bodies was evaluated by avidin–biotin–peroxidase complex (ABC) method and TUNEL assay, respectively. Percentage of Ki-67 positive cells and apoptotic bodies was calculated. The significant highest percentage of Ki-67 positive cells and apoptotic bodies was observed at mid diestrus. Positive correlation was found mainly in the alveolar epithelium between progesterone levels and percentage of Ki-67 positive cells, as well as between progesterone levels and apoptosis. In conclusion, the results from the present study suggested that progesterone may influence on proliferation and apoptosis in canine mammary tissue.