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.

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.

L-carnitine Supplemented Extender Improves Cryopreserved-thawed Cat Epididymal Sperm Motility.

Cryopreservation of epididymal sperm is an effective technique to preserve genetic materials of domestic cats and wild felids when they unexpectedly die. However, this technique inevitably causes detrimental changes of cryopreserved-thawed spermatozoa, for example, by physical damage and excessive oxidative stress. L-carnitine is an antioxidant that has been used to improve sperm motility in humans and domestic animals. This study aimed to investigate the effects of L-carnitine on cat epididymal sperm quality following cryopreservation and thawing. After routine castration, cauda epididymides were collected from 60 cat testes. The epididymal spermatozoa from 3 cauda epididymides were pooled as 1 replicate. Spermatozoa samples (16 replicates) were examined for spermatozoa quality and then randomly divided into 4 groups: 0 mM L-carnitine (control), 12.5 mM, 25 mM and 50 mM L-carnitine. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, plasma membrane integrity, DNA integrity and acrosome integrity were evaluated. The 25 mM L-carnitine significantly improved sperm motility compared with a control group (p<0.05), although this was not significantly different among other concentrations. In conclusion, supplementation of 25 mM L-carnitine in freezing extender improves cauda epididymal spermatozoa motility. The effects of L-carnitine on the levels of oxidative stress during freezing and thawing remains to be examined.

Birth of kittens after the transfer of frozen-thawed embryos produced by intracytoplasmic sperm injection with spermatozoa collected from cryopreserved testicular tissue.

The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection (ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post-ICSI, presumptive zygotes/cleaved embryos were treated with 10 µm forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/hCG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm.

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.

Intergeneric somatic cell nucleus transfer in marbled cat and flat-headed cat.

Intergeneric nucleus transfer (ig-NT) is a promising technique to produce offspring of endangered species. The objectives of this study were to (1) investigate the in vitro development of marbled cat (MC; Pardofelis marmorata) and flat-headed cat (FC; Prionailurus planiceps) ig-NT embryos reconstructed from domestic cat (DC; Felis catus) oocytes (Experiment 1), (2) evaluate the effect of individual FC donor cell lines on NT success (Experiment 2), and (3) assess the developmental ability of FC-cloned and DC-IVF embryos in vitro and in vivo after oviductal transfer (Experiment 3). In Experiment 1, the morula rate of FC-reconstructed embryos was significantly higher than those of MC and DC embryos but lower than that of parthenogenic DC embryos. However, blastocyst rate was not different. In Experiment 2, FC-ig-NT embryos reconstructed from female muscular tissue had significantly higher morula rate in comparison with those derived from other donor cell lines. However, there was no difference in blastocyst rate among cell lines. In Experiment 3, in vitro development of FC-ig-NT embryos was lower than that of DC-IVF embryos. The competency of in vivo development of FC-ig-NT and/or DC-IVF embryos was investigated by assessing pregnancy rate after their transfer into DC recipients. Domestic cat recipients receiving only FC-ig-NT embryos, FC-ig-NT embryos in one side of the oviduct and DC-IVF embryos contralaterally (co-transfer), and only DC-IVF embryos were observed. No pregnancy was detected in all recipients receiving FC-ig-NT embryos. One recipient receiving co-transferred embryos became pregnant, then delivered DC-IVF dead fetuses (n=2) and live kittens (n=6). All recipients receiving DC-IVF embryos became pregnant, and three of six recipients delivered five DC-IVF kittens. These results illustrate the developmental capacity of MC- and FC-ig-NT embryos up to the blastocyst stage. Individual donor cell line affects the developmental success up to the morula stage of FC-ig-NT embryos. Improving the developmental competence and quality of FC-ig-NT embryos may be required for implantation and development to term of FC-ig-NT offspring.

Effects of thawing temperature and post-thaw dilution on the quality of cat spermatozoa.

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70 degrees C) and to investigate the effects of post-thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25-ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37 degrees C for 15 s, (ii) at 37 degrees C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70 degrees C for 6 s, (iv) at 70 degrees C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR-14/EthD-1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 +/- 10.7 (mean +/- SD), a score of progressive motility of 4.0 +/- 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 +/- 12.1 and intact acrosome of 44.8 +/- 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70 degrees C and post-thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post-thaw incubation.

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen.

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.