Abstract This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long‐term chilled preservation. Semen samples were stored in M odena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5° C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5‐ SM and 15‐ SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks ( P < 0.05), and the 7.5‐ SM group showed improved motility indices even after 4 weeks ( P < 0.05). In vitro fertilization using spermatozoa in the 7.5‐ SM and 15‐ SM groups stored at 5° C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group ( P < 0.05), and the 7.5‐ SM group showed similar rates of fertilization and blastocyst formation in the fresh non‐stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5‐ SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5° C in a M odena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5° C for 2 weeks.
Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P < 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes.
The cooling of mammalian oocytes to sub-physiological temperatures is widely known to affect their viability through the induction of various abnormalities at all stages of meiosis.This study was to compare the kinetics of nuclear status and oocyte damage in porcine, bovine and feline ovaries stored at 4 degrees C for 5 days.The nuclear status and oocyte quality during storage were evaluated before and after maturation culture.The cold storage of ovaries decreased the proportions of porcine and bovine oocytes that remained at the germinal vesicle stage before maturation culture. The maturation rates of oocytes decreased with increasing storage time, independent of species. None of the porcine oocytes reached metaphase II (MII) after 1 day of storage. In contrast, bovine and feline oocytes from ovaries that were stored for 2 days and 3 days reached MII. DNA fragmentation in porcine oocytes from ovaries stored for 1 day was significantly higher than that in bovine and feline oocytes.The maturation competency of oocytes after the cold storage of ovaries could be related to the meiotic resumption of oocytes during storage and the occurrence of DNA fragmentation in oocytes during maturation culture.
Heat stress can engender various disorders in reproductive functions such as impairment of oocyte maturation, fertilization, and embryonic development. Astaxanthin, an extremely common carotenoid, is a typical fat-soluble antioxidant that scavenges ROS and blocks lipid peroxidation. Moreover, astaxanthin has been shown to improve the development of embryos exposed to heat stress by a reduction in stress-inducible genes. This study was conducted to investigate the effects of astaxanthin supplementation on the meiotic competence, fertilization, and development of porcine oocytes exposed to high temperature (41°C) during maturation culture. Cumulus–oocyte complexes (COC) collected from ovaries were transferred into maturation medium supplemented with astaxanthin (0, 0.25, 0.5, or 1.0 ppm) and were then cultured for 46 h at 41°C or 38.5°C. After maturation culture, the COC were subjected to IVF and embryo culture to evaluate the fertility and development of oocytes. The total cell number and DNA fragmentation in the blastocysts were assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling and Hoechst 33342 staining. The total numbers of oocytes matured at 41°C and 38.5°C in each treatment group were 432 to 470 and 426 to 444, respectively. Data were analysed using ANOVA, followed by Fisher's protected least significant difference test. Exposure to elevated temperatures during maturation culture significantly reduced the proportions of oocytes that reached metaphase II. When the COC were cultured in the maturation medium supplemented with 0.5 and 1.0 ppm of astaxanthin under heat stress conditions (41°C), the supplementation of astaxanthin significantly improved the proportions of maturation, fertilization, and blastocyst formation compared with the control group (0 ppm) (50–52%, 45–55%, and 11–12% v. 17, 25, and 6%, respectively). The supplementation of the maturation medium with 0.25 ppm of astaxanthin improved only blastocyst formation (9.6%). Similarly, the supplementation of astaxanthin at 1.0 ppm improved the proportions of maturation, fertilization, and blastocyst formation of oocytes matured at 38.5°C s compared with the control group (67, 57, and 18% v. 48, 33, and 12%, respectively). However, no beneficial effect of astaxanthin supplementation was found in the total cell number or DNA fragmentation in the blastocysts, irrespective of culture temperature. Our findings show that the supplementation of astaxanthin to maturation medium improves maturation, fertilization, and embryo development of porcine oocytes exposed to heat stress during maturation culture.
Follicle-stimulating hormone (FSH) alone can induce oestrus in bitches, but few reports describe oestrous induction by FSH because pregnant mare serum gonadotrophin (PMSG) has been more successful than FSH for oestrus induction. Real-time ultrasonography can show canine ovarian follicle development, but no method can determine or predict ovulation accurately. Moreover, the ovary location and size complicate imaging. Using ultrasonography, we investigated FSH treatment stimulation of canine ovary follicles, with clamping of the ovaries at a subcutaneous site. Bilateral malacotomy of four 5-year-old Beagle bitches (mean weight 10.3 ± 2.0 kg) with normal oestrous cycles was done using a ventral flank abdominal approach with routine techniques and materials. Each ovary that maintained blood circulation from the suspensory ligament was clamped at a subcutaneous site through muscles of the abdomen. After about six months of bilateral malacotomy, four bitches at the anestrous (two bitches) and diestrous (two bitches) stages of the oestrous cycle were given 0.5 Armour units of FSH twice daily for 5 days. Examinations with ovarian ultrasonography with 7.5 MHz sector transducer, vaginal cytology, and serum concentrations of progesterone and oestradiol were performed daily from the day before the start of FSH treatment through 7 days after FSH treatment. After 15 days of ovarian examination, each bitch received the same FSH treatment twice continually at 15-day intervals. No serosanguineous vaginal discharge was observed during the ovarian examination. The concentrations of progesterone (<0.045–9.6 ng mL–1) and oestradiol (<9.7–81.4 pg mL–1) varied through all treatments. Comparison of the concentrations of progesterone (<0.045–7.6 ng mL–1) and oestradiol (<9.7–30.3 pg mL–1) at the start of FSH administration in each trial revealed that elevated concentrations of both progesterone and oestradiol were observed in the first treatment in 3 bitches. Regarding the second and third treatments, no elevation of concentration was found for progesterone or oestradiol. A new follicular growth was observed in 1 animal after the third FSH treatment, but no follicular growth was found for the other animals. No correlation was found between follicular development and the profile of either progesterone or oestradiol. Ultrasonography proved that FSH stimulation alone cannot induce follicular growth by a single treatment, but it might increase the levels of progesterone and oestradiol, which are not correlated with follicular development and oestrous cycles at the start of FSH treatment.
Liquid preservation can be used as an alternative to freeze-thawing for preserving semen for AI. The efficiency of some boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the viability and penetrability of boar spermatozoa preserved at 5°C in a modified Modena-based extender supplemented with either 100 μM vitamin C (Vc), 100 μM vitamin E (Ve), or 100 μM Vc + 100 μM Ve (Vc + e). The final sperm concentration was adjusted to cells mL–1 and the semen was then stored at 5°C for 4 weeks. In Experiment 1, the semen samples were assessed every week during the 4-week storage in each extender for the following factors: motility, by using computer-assisted semen analysis (CASA); viability, by using the Live/Dead fluorescence viability assay; plasma membrane integrity, by using the hypoosmotic swelling test (HOST); and acrosome integrity, by using fluorescein isothiocyanate (FITC)-labelled peanut agglutinin staining. In Experiment 2, we examined the penetrability of spermatozoa that had been stored in each extender for 4 weeks and the development of fertilized oocytes. Data were analysed using ANOVA. In Experiment 1, when the semen was stored for 2 weeks, the mean percentage values of total sperm motility and viability for semen stored with Ve were significantly higher than those for semen stored without Vc and Ve (control group) (84.3 vs 67.9% and 59.8 vs 51.2%, respectively; P < 0.05). Moreover, the percentage sperm motility for semen stored for 4 weeks tended to be higher in the Ve group than in the control group (44.2 vs 32.7%; P < 0.1). Storage with Vc or Vc + e did not improve sperm motility and viability of semen. The plasma membrane integrity and acrosome integrity of semen did not significantly differ among the groups during the 4-week storage. In Experiment 2, the rates of sperm penetration and of development to blastocysts of fertilized oocytes did not differ between the Ve and control groups (33.0 vs 28.5% and 14.9 vs 10.1%, respectively; P > 0.05). However, storage with Vc reduced the rate of oocyte development compared with the Ve and control groups (1.1%; P < 0.05). In conclusion, adding Ve to the semen extender may improve the motility and fertility of boar semen stored at 5°C. However, adding Vc has a harmful effect on the quality and fertility of stored boar semen.
Follicular growth in bitches is usually detected indirectly through behaviour observation, vaginal smears, and hormonal assay in blood. Although real-time ultrasonography can reveal the development of canine ovarian follicles, no method has been established to determine or predict ovulation accurately. Moreover, the location and small size of the ovaries make imaging technically difficult. This study was conducted to investigate follicular waves of canine ovaries stimulated by hormone treatment, in which ovaries had been clamped at a subcutaneous site. Bilateral malacotomy of 3 bitches (4 years of age) at the anestrous (2 bitches) and proestrous (1 bitch) stages of the oestrous cycle was performed using a ventral flank abdominal approach with routine techniques and materials. Each ovary that maintained blood circulation from the suspensory ligament was clamped at a subcutaneous site through muscles of the abdomen. Oestrus was induced using subcutaneous administration of 500 IU of eCG and 1000 IU of hCG (eCG/hCG). Each bitch was given 1000 IU of hCG at 11 days after eCG/hCG administration. Examinations with ovarian ultrasonography using a 7.5-MHz sector transducer, vaginal cytology, and serum progesterone assay were performed daily until 20 days after eCG/hCG administration, and every 10 days thereafter from 20 days to 60 days. Serosanguineous vaginal discharges and vaginal cytology of 2 of the bitches were observed. Follicular growth (>1.1 mm in diameter) was observed in all bitches after eCG/hCG administration. The appearance of new follicular growth was observed on 2 days, 6 days, and 8 days after eCG/hCG administration. The mean diameter of follicles reached 4.3 to 5.5 mm, and the maximum numbers of follicles in bitches were 11 to 16. However, all follicles regressed, irrespective of hCG administration. Elevation in progesterone levels (>2 ng mL–1) after eCG/hCG administration was observed from 2 days to 12 days after eCG/hCG administration. No correlation was found between follicular development, progesterone profiles, and vaginal smear characteristics. Follicular growth clamped at the subcutaneous site can be monitored easily using ultrasound without an experienced operator. Moreover, ultrasonography proved that hormonal stimulation can induce follicular growth, but the day of appearance of new follicles varied.
Unlike the oocytes of other species, cat oocytes have a unique characteristic in that they can mature in vitro after temporary storage at 5°C. Temporal storage of ovaries can aid in preserving oocytes from the ovaries of endangered felids that sometimes die suddenly in the field. The present study was conducted to evaluate the effects of the duration of cold storage of cat ovaries on the quality and meiotic competence of their oocytes. Domestic cat ovaries were collected from sexually mature queens (40 ovary pairs) at various stages of the reproductive cycle, after routine ovariohysterectomy performed at local veterinary clinics. After excision, the ovaries were stored at 5°C in physiological saline for 0, 1, 2, 3, 4 and 5 days until oocyte recovery. The cumulus–oocyte complexes with uniform and dark-pigmented ooplasm and more than 2 layers of cumulus cells were collected (n = 526) and then cultured for 24 h in maturation medium. This maturation medium consisted of tissue culture medium 199 supplemented with 4 mg mL–1 of bovine serum albumin, 0.1 IU mL–1 of human menopausal gonadotropin, 10 IU mL–1 of hCG, 1 μg mL–1 of 17 β-oestradiol and 50 μg mL–1 of gentamicin. The status of meiosis and fragmented chromatin in the oocytes after the 24-h maturation culture was assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling and Hoechst 33342 staining. Data were analyzed using analysis of variance. The percentage of oocytes that reached metaphase II was greater for ovaries kept without cold storage (control group; 60.5%) than for ovaries kept for 1, 2, 3, 4 and 5 days in cold storage (maturation of 28.6, 38.3, 23.2, 1.6 and 2.2%, respectively; P < 0.05). In the cold storage group, the maturation rates (1.6–2.2%) of oocytes from ovaries stored for more than 4 days were significantly lower (P < 0.05) than those (23.2–38.3%) of oocytes from ovaries stored for less than 3 days. Moreover, oocytes with fragmented chromosomes after the maturation culture were not observed in the case of the control group, whereas 24.4 to 51.8% of the oocytes in the cold storage group had fragmented chromosomes, irrespective of the storage period. These results indicate that cold storage of cat ovaries decreases the meiotic competence of oocytes and influences the quality of oocytes matured in vitro.
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