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

In vitro Survival of Follicles Collected from Domestic Cats&#8217; 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

Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems

The aim of this study was to evaluate the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of oocytes obtained from bitches at different reproductive stages (follicular, luteal, anoestrous). In System A (control) oocytes were matured for 72h in base medium (BM) with 10IUmL-1 human chorionic gonadotrophin (hCG), 1?gmL-1 progesterone (P4) and 1?gmL-1 oestradiol (E2); in bi-phasic System B oocytes were matured for 48h in BM with hCG and for 24h in BM with P4; in bi-phasic System C oocytes were matured for 48h in BM with hCG, P4 and E2, and for 24h in BM with P4; in System D, oocytes were cultured in BM without hormonal supplementation. Data were analysed by ANOVA. There was a positive effect of the bi-phasic systems on germinal vesicle breakdown, metaphase I and metaphase II rates, irrespective of reproductive status (PP<0.001). The stage of the oestrous cycle did not influence maturation rates

Canine and feline oocyte lipid profile by Matrix-Assisted Desorption Ionization time-of-flight Mass Spectrometry (MALDI-TOF MS)

OBJECTIVES AND METHODS. Canine and Feline oocytes present an elevate cryosensibility, mostly due to their high lipid content (1). It is believed that the presence of intracellular lipid droplets could be responsible for uneven intracellular ice formation which could affect the freezing-thawing process (2). Studies on the lipid composition of oocytes from these species may contribute with the improvement of the cryopreservation process as well as to the advancement of in vitro culture conditions. Thus, the aim of this work is to apply the technique of matrix-assisted desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to obtain the lipid profiles of single canine and feline oocytes and compare with the lipid profiles obtained for the bovine oocytes, a species commonly used for cryopreservation studies. Ovaries were collected from three diestrous bitches (various breeds, age 1-7 y) and one queen by routine ovariohysterectomy and sliced in PBS-PVA to release the cumulus-oocyte complexes (COCs). Oocytes were graded according to morphological characteristics and only grade I COCs were selected and stored in PBS solution at -20\uf0b0C until analysis. For MALDI-TOF MS analysis, samples were washed in H2O/MeOH 1:1 (v/v) and placed (1 to 3 oocytes/spot) in the MALDI target plate. Samples were then covered with 2.5 dihydroxybenzoic acid (DHB) as the organic matrix. A Synapt HDMS (Waters Corp. Milford, MA, USA) equipped with a MALDI source was used for the experiments. All mass spectra were manually collected for around 1 min in the positive ion mode, at the m/z range of 700-1000. Mass spectra collected from individual embryos were processed using the software MassLynx 4.1 (Waters Corp. Milford, MA, USA). RESULTS. This is the first report about lipid fingerprint of canine and feline oocytes by MALDI-TOF MS. The technique has been already reported for bovine oocytes and embryos, as well as human and sheep oocytes (3). Besides the analysis workflow simplicity (no lipid extraction), intact complex lipid species such as sphingomyelins (SM), phosphocholines (PC) and triacylgycerols (TAG) can be detected because lipids are easily ionizable by MALDI using DHB as the matrix. By gas chromatography (GC), traditionally used for lipid analysis in gametes and embryos, only fatty acyl residues can be detected and structural information of lipids is lost. Mass spectra of bitch and queen oocytes were similar and characterized mainly by intense ions correspondent to the m/z values of PC containing 34 carbons (of m/z 780.7 to 786.7) and 36 carbons (of m/z 808.7 to 812.7), but mainly PC (34:1) of m/z 782.8 (sodiated PC 34:1), m/z 723.7 (fragment of PC 34:1) and 760.8 (protonated PC 34:1), which is the main phospholipid present in the cellular membrane and this fact has been also observed for bovine oocytes (3). Regarding the TAG species, m/z values correspondent to species containing 52 and 54 carbons were more prominent in canine and especially in feline oocytes, compared to the bovine (3). CONCLUSIONS. Lipid attributions described herein must be confirmed by MS/MS experiments, but our data indicate that MALDI-TOF MS is able to detect the presence and the chemical structure (number of carbons present in the fatty acyl residues and unsaturations) of PC and TAG species in canine and feline oocytes. We envisage MALDI-TOF MS will become a relevant tool in studies aiming at improving canine and feline oocyte cryopreservation success. (1) -Luvoni GC, Pellizzari P, Battocchio M. Effects of slow and ultrarapid freezing on morphology and resumption of meiosis in immature cat oocytes. J Reprod Fertil Suppl 1997; 51: 93-98. (2) Luvoni GC. Gamete cryopreservation in the domestic cat. Theriogenology 2006; 66: 101-11. (3) Ferreira CR, Saraiva SH, Catharino RR, Garcia JS, Gozzo FC , Sanvido GB, Santos LFA, Lo Turco EG, Pontes JHF, Basso AC, Bertolla RP, Sartori R, Guardieiro MM , Perecin F , Meirelles FV , Sangalli JR, Eberlin MN. Single embryo and oocyte lipid fingerprinting by mass spectrometry. Journal of lipid research 2010; 51: 1218-1227