Hormonal profile and follicular dynamics concurrent with CIDR and insulin modified Ovsync TAI programs and their impacts on the fertility response in buffaloes.

Fifty one cyclic Egyptian buffaloes were used to study the hormonal profile and follicular dynamics concurrent with CIDR and insulin modified Ovsync TAI programs and their impacts on the consequent fertility responses. The buffaloes were randomly assigned into 3 ovulation synchronization protocols: Ovsync-alone (n = 13, control) CIDR-sync (n = 20) and Insulin-sync (n = 18). Ovsync-alone protocol consisted of two im injections of 20 µg bueserlin (GnRHa) on Day 0 (GnRH 1) and on Day 9 (GnRH 2) with an im injection of 500 µg of cloprostenol sodium (PGF2 α) on Day 7. The CIDR-sync protocol consisted of the same treatment protocol as in Ovsync in addition to intra-vaginal insertion of CIDR (contains 1.38 gm of progesterone) on Day 0 followed by removal on Day 7. The Insulin-sync protocol consisted of the same treatment protocol as in Ovsync plus 3 sc injections of insulin at a dose of 0.25 i.u/1 kg, on Days 7, 8, and 9. Buffaloes in all groups were inseminated 16 h after GnRH2 by the same inseminator using frozen semen in straws. Blood samples were collected on Days 0, 3, 5 for serum progesterone assay and on Day 9 to measure serum concentrations of estradiol, insulin and IGF-1. Transrectal ultrasonographic scanning of the ovaries was conducted on Days 7, 8 and 9 to record the diameter of the largest follicle. Pregnancy diagnosis was conducted on Day 30 post-TAI by trans-rectal ultrasonographic scanning of the uterus to calculate conception rate. The serum progesterone concentration showed an increase (p < 0.01) in pregnant compared with non-pregnant buffaloes in both Ovsync-alone and Insulin-sync groups, but not in CIDR-sync group (p > 0.05) on Days 3 and 5. The serum estradiol concentration on Day 9 showed an increase (p < 0.01) in pregnant compared with the non-pregnant buffaloes in all of the treated groups. In Insulin-sync and Ovsync-alone groups, the diameter of the largest follicle (LF) was larger (p < 0.01) in pregnant compared with non-pregnant buffaloes, but in CIDR-sync, the diameter of the (LF) was larger (p < 0.01) in non-pregnant compared with pregnant buffaloes. Also, the results showed that the greatest diameter of LF was observed in pregnant buffaloes in Insulin-sync compared with either pregnant or non-pregnant buffaloes in all groups. It is concluded that modified CIDR-sync and Insulin-sync could improve fertility response through modulating hormonal profile and follicular dynamics in buffaloes during low breeding season.

Mitochondrial aggregation patterns and activity in in vitro cultured bovine oocytes recovered from early antral ovarian follicles.

The low developmental competence seen in in vitro cultured oocytes collected from early antral follicles may be related to their mitochondrial status. The aim of this study was to examine the chromatin configuration, pattern of mitochondrial aggregation and mitochondrial activity of non-cultured and in vitro-cultured bovine oocytes originating from early antral ovarian follicles. Cumulus-oocyte complexes with adjacent granulosa cells (COCGs) were recovered from early antral follicles of 0.4 to 0.8 mm diameter. Control (Day 0) oocytes were recovered from freshly collected COCGs and fixed and stained. Selected COCGs were placed in growth culture for 7 days (Day 7) or 14 days (Day 14). Following growth culture, COCs with normal appearance were placed in maturation medium (IVM) for 24 h and then fixed and stained with MitoTracker CMTM Ros Orange and Hoechst 33258. The percentage of oocytes with an immature meiotic configuration after growth culture decreased with the time of growth culture, being 96.7; 72.5 and 35.4% respectively for Day 0, Day 7 and Day 14 of culture; the remaining oocytes were degenerating or resuming meiosis. After subsequent IVM the highest proportion of oocytes in diakinesis or metaphase I was found in the D7+IVM group (59.4%). When growth culture was prolonged to day 14 and IVM, the number of degenerated oocytes increased dramatically after IVM. The mitochondrial distribution in the oocytes changed from homogeneous to heterogeneous as growth culture time increased. The respiratory activity as measured by fluorescence intensity increased over the time of growth culture, and was highest in oocytes that had resumed GVBD. In conclusion, for oocytes in isolated COCGs from early antral follicles, culture conditions longer than 7 days should be more adapted for a slow nuclear maturation accompanied by a decreased energy metabolism to prevent chromatin pycnosis.

Holding bovine oocytes in the absence of maturation inhibitors: kinetics of in vitro maturation and effect on blastocyst development after in vitro fertilization.

Holding immature oocytes before maturation simplifies the transport of oocytes and aids in scheduling later manipulations. We examined the effect of holding bovine oocytes in the absence of meiotic inhibitors on their subsequent meiotic and developmental competence. Oocytes were matured immediately after recovery (control) or were held in a mixture of 40% TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts, and 20% FBS, at room temperature for 16 to 18 h (EH-held) and then matured. Chromatin status was determined at 0, 10, 14, 18, and 22 h of maturation culture. Oocytes were fertilized in vitro after either 18 or 22-24h maturation. The EH treatment maintained oocytes at the germinal vesicle stage (79.3%, vs. 87.7% for control oocytes at 0 h; P>0.05). Upon culture, held oocytes matured more quickly than did control oocytes. The proportions of mature oocytes were not significantly different between groups at 18 h (EH-held, 80.6% and control, 79.3%); however, after 22 h significantly more EH-held than control oocytes had degenerated (24.1% vs. 4.5%, P<0.0001). Blastocyst development was similar between groups for oocytes fertilized after 18 h maturation (EH-held, 29.6% and control, 27.8%). When oocytes were fertilized after 22-24h maturation, EH-held oocytes yielded lower blastocyst development than did control oocytes (16.5% vs. 29.3%, P<0.05). In conclusion, bovine oocytes may be effectively held in the EH treatment before maturation without adversely affecting meiotic or developmental competence. However, holding affects the kinetics of maturation and this must be taken into account when subsequent manipulations are performed.

Endocrine, morphological, and cytological effects of a depot GnRH agonist in bovine.

The present study was conducted to assess effects of the gonadotropin-releasing hormone agonist (GnRHa) triptorelin in dairy heifers. The peptide was released from a commercial 4-week depot formulation (Decapeptyl Depot) administered at animals' estrus (day 0). First experiment (EXP I, n=5), which was aimed to explore the availability of peptide, detected a maximum of triptorelin concentration between day 2 and 5 after depot injection, and the peptide remained detectable by RIA in peripheral blood for about 3 weeks. In further experiments, the peptide release was terminated on day 9 (EXP II, n=16) or day 21 (EXP III, n=47). Treatment effects were studied on follicular development, the characteristics of cumulus-oocyte complexes (COCs) (EXP II; EXP IIIa) and secretions of LH and progesterone (EXP IIIb). Results showed that the occurrence of the pre-ovulatory LH surge was more uniform in treated heifers than that in controls. The duration of ovulation periods was similar amongst the heifers of EXP II, but more compact amongst those of EXP III each compared with the respective controls. Post-ovulatory, the number of LH pulses was significantly reduced by treatment, whereas both basal LH and progesterone concentrations were elevated on a few days. Follicular growth was reduced only by the prolonged influence of the GnRHa. There were increased proportions of both degenerated COCs and immature oocytes from small follicles (<3mm in diameter), and meiotic configuration and quality of oocytes isolated from follicles 3-5mm were changed after the prolonged, 21-day treatment. These results indicate that a continuous influence of a GnRHa over more than 1 week may increasingly impair the development of bovine follicles and oocytes. This may have some significance for the development of novel GnRH-based techniques in regulating the reproductive function in cattle.

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro.

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.

Dynamics of meiosis and protein kinase activities in bovine oocytes correlated to prolactin treatment and follicle size.

Oocyte developmental competence depends on the size of the original follicle and is affected by compounds like prolactin. We wished to investigate nuclear and cytoplasmic maturation of bovine oocytes correlated to their origin and response to prolactin treatment, by monitoring at frequent intervals meiotic configuration of chromosomes and activity of histone H1 and MAP-kinase. Bovine ovaries were obtained from a slaughterhouse and oocytes were recovered by follicle isolation. Oocytes (n = 1,397) with a compact cumulus were selected from small (2 to 3 mm) and large (4 to 5 mm in diameter) follicles and cultured up to 28 h in TCM 199+20% bull serum with or without 50 ng/mL bovine prolactin. Four groups of oocytes were formed: originating from small or large follicles, and treated or not treated with prolactin. At the scheduled time intervals for in vitro maturation, cumulus oocyte complexes from the 4 groups were randomly selected and the oocytes were analyzed for histone H1 and MAP-kinase, and for chromatin configuration. The first meiotic division took longer to complete in oocytes from large follicles (P < 0.01). Under the influence of prolactin the meiosis was prolonged in oocytes both from small and large follicles (P < 0.05). Histone H1 and MAP-kinases started to be activated at approximately the same time, around 6 h after beginning maturation. But after this time, significantly lower levels of both kinase activities were found in oocytes treated with prolactin, especially those treated during Meiosis I (P < 0.05). Our results indicate a correlation of chromatin configuration and histone H1/MAP-kinase activities.