Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of \u3b1-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of \u3b1-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) \u2013 epididymal sperm were frozen with a commercial Botucrio\uae extender; group 0.3, group 0.6 and group 0.9 \u2013 the extender was supplemented with 0.3, 0.6 and 0.9 mM of \u3b1-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three \u3b1-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the \u3b1-tocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of \u3b1-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of \u3b1-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages

In vitro Survival of Follicles Collected from Domestic Cats’ Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1

Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 \ub1 22.1 lm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 \ub1 14.4 lm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 \ub1 15.9 lm, 63.6%, respectively) than that cultured without IGF-1 (26.7 \ub1 14.4 lm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase

In vitro survival of preantral follicles recovered from queens at different stages of estrous cycle and cultured with IGF-1

OBJECTIVE AND METHODS. In vitro growth of preantral follicles may supply high numbers of competent oocytes that can be destined to in vitro embryo production. However, optimal culture systems that support follicular and oocyte development in vitro have not yet been defined. Ovarian follicular development is regulated mainly by endocrine, autocrine, and paracrine factors and follicles are exposed to specific hormonal environments during the different stages of the estrous cycle. The intraovarian insulin-like growth factor-I (IGF-1) regulates in vivo follicular development and there are important evidences that it stimulates in vitro growth of preantral follicles in domestic animals (1). Feline preantral follicles have been previously cultured in vitro (for review 2) and it has been suggested that IGF-1 enhances oocyte metabolism (3). However, no information are available on the response of preantral follicles recovered in different phases of the estrous cycle to cultural conditions. Thus, the aim of this study was to investigate the in vitro survival of preantral follicles recovered from ovaries of queens in follicular or luteal phase of the estrous cycle and cultured in presence of IGF-1. Twelve queens were housed with 12 hours of light and submitted to estrous induction with IM injection of 100 UI eCG (Novormon\uae- Intervet) and 100 UI hCG (Vetecor\uae- Hertape Calier) 82 hours later (4). Six queens were spayed 96 h after eCG administration (follicular phase), the others 36 h after the hCG injection (luteal phase). Estrous phases were confirmed by vaginal cytology prior to the surgical procedure and macroscopic evaluation of ovarian structures after excision. Preantral follicles surrounded by complete basal membrane and containing more than one layer of granulosa cells were retrieved from excised ovaries and selected as previously described (5). A total of 72 follicles were cultured for 6 days at 38.5 \u2daC and 5% CO2 in air in Minimum Essential Medium (Sigma Chemical Co., USA) with IGF-1 100 ng/ml (Sigma) or without (control). Before and after culture, follicular diameter was recorded and follicular viability was assessed by fluorescein diacetate (FDA, Sigma) staining. Increase (%) in diameter was analyzed by Tukey\u2019s and Fisher\u2019s test, viability rates by Chi-square test (P<0.05). RESULTS. After 6 days of culture, preantral follicles retrieved during follicular phase showed a significant increase in the size and a higher viability rate than those retrieved in the luteal phase of the estrous cycle (18.8% vs.11.5%; P= 0.0001 and 75% vs. 62.5%; P=0.004). However, when IGF-1 was added to the culture medium, follicles retrieved in follicular or luteal phase showed similar increase in diameter (14.9% vs.13.4%; P>0.05) and viability (73.8% vs. 76%; P>0.05). Regardless of the stage of the estrous cycle, overall results showed that the increase in diameter was not different in follicles cultured with or without IGF-1 (14.6% vs. 15.8%; P>0.05), but follicular survival was enhanced when IGF-1 was added to the culture medium (75% vs. 69.4%; P=0.0001). CONCLUSIONS: Present data suggest that in vitro survival of preantral follicles is affected by the estrous stage of the donor and IGF-1 improves survival of follicles retrieved in luteal phase of the estrous cycle. Thus, the hormonal environment of the follicles within the ovary might impact their potential development when isolated and cultured. Further investigations on growth factors are needed to evaluate their effect on follicles with reduced in vitro developmental capability. REFERENCES (1) Giudice LC. Insulin-like growth factors and ovarian development. Endocr Rev 1992; 13: 641-669. (2) Jewgenow K, Paris MCJ. Preservation of female germ cells from ovaries of cat species. Theriogenology 2006; 66: 93-100. (3) Jewgenow K. Impact of peptide growth factors on the culture of small preantral follicles of domestic cats. Theriogenology 1996; 45: 889-895. (4) Villaverde AI, Melo CM, Martin I, Ferreira TH, Papa FO, Taconeli CA, Lopes MD. Comparison of efficiency between two artificial insemination methods using frozen-thawed semen in domestic cat (Felis catus): artificial insemination in domestic cats. Anim Reprod Sci 2009; 114: 434-442. (5) Lima AK, Bezerra MB, Oliveira LC, Figueiredo JR, Silva LDM. Isolamento e caracteriza\ue7\ue3o de fol\uedculos ovarianos pr\ue9-antrais em gatas dom\ue9sticas (Felis catus). Rev Bras Reprod Anim 2003; 27: 396-397