Serum concentrations of melatonin in prepubertal gilts exposed to either constant or stepwise biweekly alteration in scotophase

Melatonin (MEL), a hormone known to mediate photoperiodic cues, is secreted from the pineal gland in a circadian fashion in numerous species. The transduction of photoperiodic information into the secretion of MEL, however, remains controversial in the pig. To determine whether domestic pigs have a nocturnal increase in serum melatonin when exposed to equatorial photoperiods only, 24 prepubertal gilts (38.7 ± 0.7 kg; 104.5 ± 0.8 d) and 12 mature ewes, serving as positive controls, were assigned randomly to one of two environmentally regulated rooms. The light (L):dark (D) schedule in one room remained constant (10 L:14 D), while the other room scotophase (darkness duration) was decreased by 1 hr every 2 wk (Experiment 1). After a 2-wk acclimation to each new schedule, 6 ewes and 6 gilts in each room were bled by venipuncture at 2-hr intervals for 22 hr. Experiment 2 was conducted as described for Experiment 1, except that the LD schedule in one room remained constant (15L : 9D) while length of scotophase in the other room was increased by 1 hr every 2 wk. In gilts that were exposed to constant 10L:14D, scotophase MEL in serum averaged 103 ± 13 pg/ml as compared with 57 ± 13 pg/ml in the photophase. Using each gilt's initial photophase value as a statistical covariate, scotophase MEL in the constant 10L:14D schedule was higher (P < 0.001) than photophase MEL. A similar analysis of MEL in gilts exposed to stepwise biweekly decreases in scotophase revealed a scotophase elevation (P < 0.05) in only certain LD schedules (i.e., 12L: 12D and 13L:11D) but the same trend was present throughout all LD schedules. Subjective examination of individual gilt profiles revealed that 56% of gilts had a nocturnal increase in serum MEL in Experiment 1. However, only 10% of the MEL profiles were closely coupled to the environmental LD periods. Overall, mean serum MEL was slightly elevated during scotophase in gilts, but the nocturnal elevation of MEL in gilts was of lesser magnitude and more variable than in ewes. Data from these two experiments suggest that the domestic pig has an inherently weak nocturnal elevation in serum MEL, and the ability to detect these rises is dampened by considerable pig-to-pig variability.     

Overfeeding and underfeeding have detrimental effects on oocyte quality measured by in vitro fertilization and early embryonic development in sheep

To determine effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 48) were divided into control, overfed (ad libitum feeding), and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Follicular development was induced by twice-daily injections of FSH on days 13 and 14 of the estrous cycle, and ovaries and blood samples were collected on day 15 of the estrous cycle. During the 8-wk experiment, for control ewes BW and BCS did not change, but for overfed ewes mean (± SEM) BW and BCS increased (11.8 ± 1.1 kg and 2.0 ± 0.1, respectively) and for underfed ewes decreased (14.2 ± 0.9 kg and 0.7 ± 0.1, respectively). The number of follicles was determined; oocytes were collected and subjected to in vitro maturation and fertilization. After IVF, developing embryos were evaluated throughout the 8-d culture period. The proportion of cleaved oocytes after IVF and developing morula and blastocyst were less (P < 0.0001) in overfed and underfed ewes than in control ewes. However, number of visible follicles, total number of oocytes, number of healthy oocytes, and percentage of healthy oocytes were similar for control, overfed, and underfed ewes. Serum insulin concentration was greater (P < 0.05) in overfed ewes than in underfed ewes, estradiol 17-β (E₂) concentration was greater (P < 0.05) in underfed ewes than in overfed ewes, but triiodothyronine (T₃) and thyroxine (T₄) concentrations were similar in all treatment groups. These data show that inadequate feeding has a negative effect on oocyte quality which results in lower oocyte cleavage after IVF and morula and blastocyst formation; overfeeding increased serum insulin and underfeeding increased serum E₂ but not T₃ or T₄. These data emphasize the importance of diet for reproductive and metabolic functions. Furthermore, the mechanisms through which enhanced or decreased energy in diet affect oocyte quality and serum insulin and E₂ concentrations remain to be elucidated.     

Bovine Oocyte Quality in Relation to Ultrastructural Characteristics of Zona Pellucida,Polyspermic Penetration and Developmental Competence

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus–oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP’s pores were evaluated in squares of 6.4‐μm width. In group B, oocytes were fertilized in vitro. After 48 h, non‐cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 ± 0.07 μm, which was smaller than the observed on oocytes of quality 2 (0.83 ± 0.10 μm), quality 3 (1.02 ± 0.22 μm) and quality 4 (1.38 ± 0.59 μm) (p ≤ 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p ≤ 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore’s diameter.     

Melatonin but not the ram-effect reactivates quiescent ovarian activity of mid-anestrous ewe

The present study was conducted to examine how the social cue emanating from rams, the ram-effect, would influence the onset of melatonin-induced reproductive activity in anestrous ewes. Twenty non-lactating ewes were randomly allocated into 4 groups as follows based on a combination of the melatonin treatment (MEL) and the ram-effect (RAM): ewes of Groups A (MEL + RAM) and B (MEL) were subcutaneously implanted with melatonin capsules on April 18 (Day 0), which increased plasma melatonin levels by about 200 pg/ml for at least 5 months, while Groups C (RAM) and D (control) were untreated with melatonin. Rams were introduced to Groups A and C on Day 0, whereas Groups B and D were isolated from rams. Ovarian function of the ewe was assessed on June 9-21 (Days 52-64) by monitoring plasma progesterone (P) profiles. Luteal function (plasma P greater than 1 ng/ml for a week or longer) was evident in all the melatonin-treated ewes but only one in those untreated: 5/5 in Group A, 5/5 in Group B, 1/5 in Group C and 0/5 in Group D. By ultrasonography on Day 105 all the Group A ewes were diagnosed pregnant but none in the Group C despite that both the two groups had been run with rams. These results indicate that chronic melatonin treatment is capable of advancing the reproductive recrudescence in seasonally anestrous ewes, and that progonadal effects of rams are subtle, if any, during the mid-anestrous period.     

N-acetyl-l-cysteine supplementation improves boar spermatozoa characteristics and subsequent fertilization and embryonic development

The effects of 1.0 mm N‐acetyl‐l ‐cysteine (NAC) supplementation during the incubation of frozen–thawed and preserved boar sperm were studied in addition to subsequent oocyte IVF. Frozen–thawed and preserved boar sperm were supplemented with 1.0 mm NAC and incubated for 60 min to allow capacitation to occur followed by the addition of calcium ionophore 23187 to induce the acrosome reaction. The number of sperm having undergone the acrosome reaction was determined using the Wells–Awa staining technique. DNA damage was detected using single‐cell gel electrophoresis. Membrane lipid peroxidation was estimated by the end point generation of malondialdehyde (MDA). Frozen–thawed sperm was not different in the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) more DNA damage (59.8 ± 1.0) compared to preserved sperm (32.0 ± 1.0%). Supplementing 1.0 mm NAC did not have an effect on the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) less DNA (39.2 ± 1.0%) damage compared to no antioxidant supplementation (52.7 ± 1.0%). Frozen–thawed sperm produced a significantly higher (p < 0.05) concentration of MDA (2.08 ± 0.05 μm MDA/10⁷ cells) compared to preserved sperm (1.82 ± 0.05 μm MDA/10⁷ cells), and non‐supplemented sperm produced a significantly higher (p < 0.05) concentration of MDA (3.62 ± 0.05 μm MDA/10⁷ cells) compared to the 1.0 mm NAC‐supplemented sperm (0.28 ± 0.05 μm MDA/10⁷ cells. Supplementation or semen storage method had no effect on IVF or embryonic development. These results indicate that supplementation with 1.0 mm NAC improved the ability to use frozen–thawed boar sperm during IVF as it reduces the DNA fragmentation and lipid peroxidation of the sperm.     

In Vitro Fertilization (IVF) in Straws and a Short Gamete Coincubation Time Improves the Efficiency of Porcine IVF

The present study was designed to evaluate three different in vitro fertilization (IVF) systems: a straw‐IVF system with 10 min of coincubation, a straw‐IVF system with 6‐h coincubation and the microdrop‐IVF system with 6‐h coincubation (the traditional IVF system used routinely in most of IVF laboratories) in an attempt to reduce polyspermic penetration ( Experiment 1 ). When the straw‐IVF system was tested in combination with two coincubation times, the use of 10 min of coincubation significantly increased (p < 0.001) the penetration rate and the efficiency of fertilization (67.7 ± 6.4% vs 31.9 ± 6.5% and 41.5 ± 2.5% vs 17.6 ± 2.5% for 10 min and 6 h, respectively), while there were no significant differences in the incidence of monospermy between both systems (64.3 ± 5.1% and 67.7 ± 3.4%, for 10 min and 6 h, respectively). The penetration rate in the 6‐h microdrop‐IVF system was higher (93.8 ± 3.6%; p < 0.001) compared with the 10‐min straw‐IVF system (67.7 ± 6.4%), however, monospermy was severely reduced (25.0 ± 4.3% vs 67.7 ± 3.4%, for the 6‐h microdrop‐IVF system and 10‐min straw‐IVF system, respectively). The efficiency of the IVF showed similar values between microdrop and 6‐h straw‐IVF systems, but efficiency was significantly improved (p < 0.05) when the 10‐min straw‐IVF system was used. Experiment 2 was designed to compare porcine in vitro embryo production in two IVF systems, the 6‐h microdrop‐IVF system (1000 sperm per oocyte) and 10‐min straw‐IVF system (30 000 sperm per oocyte). The blastocyst formation rates tended (p = 0.06) to be higher when the 10‐min straw‐IVF system was used compared with the 6‐h microdrop‐IVF system. In addition, the number of total cells per blastocyst increased significantly (p < 0.05) in the 10‐min straw‐IVF system. These results showed that the 10‐min straw‐IVF system is an effective way to decrease polyspermic penetration, and improve the efficiency of fertilization and the quality of blastocysts in terms of cell number per embryo.     

Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes

The effects of progesterone (P₄) on follicular growth and fertility in ewes were examined. In Experiment 1, 22 ewes received either one or three packets of P₄ (5 g/packed) or an empty packet subcutaneously (sc) from Days 5 to 15 of the estrous cycle (estrus = Day 0). On Day 6, P₄-treated ewes received 12.5 mg of prostaglandin F₂α. Follicles ⩾3 mm in diameter were observed via transrectal ultrasonography daily from Day 4 through estrus, corpora lutea (CL) were observed 5 to 7 d after estrus. Ewes with low (LOW; ⩽1 ng/ml; n = 5), intermediate (MED; > 1 and <2 ng/ml; n = 10), or normal (NOR; ⩾2 ng/ml; n = 7) P₄ in jugular plasma on Days 7 through 15 differed in follicular development. The largest follicle at estrus was larger in ewes with LOW vs. MED and NOR P₄ (7.8 ± 0.3 vs. 6.9 ± 0.2 mm; P < 0.05). Treatments differed in proportions of multiple-ovulating ewes, in which the oldest ovulatory follicle was first observed before Day 10 (LOW: 3 of 3, MED: 6 of 10, NOR: 0 of 5, respectively; P < 0.05). Estradiol was higher early in the treatment period in LOW ewes than in MED and NOR ewes (day × treatment; P < 0.05). In Experiment 2, ewes received 5 mg of P₄ in corn oil (low progesterone [LP]; n = 51) or 2 ml of corn oil (CON; n = 49) sc every 12 hr on Days 6 through 14 of the estrous cycle before mating. LP ewes received 15 mg of prostaglandin F₂α on Day 6. Mean serum P₄ on Days 7 through 15 was 0.6 ± 0.1 ng/ml in LP and 1.9 ± 0.1 ng/ml in CON ewes. Eleven LP and 12 CON ewes were scanned daily from Day 4 through mating, and in all ewes (n = 93), CL were counted 10 d after mating and embryos were counted at 25, 40, and 60 d of gestation. In multiple-ovulating ewes, day of cycle of appearance was earlier for the oldest (Day 6.1 ± 0.8 vs. 10.4 ± 0.8) but not second oldest (Day 11.7 ± 1.0 vs. 12.2 ± 0.9) ovulatory follicles in LP compared with CON ewes. The conception rate was lower in LP (72%) than in CON ewes (98%; P < 0.01). However, numbers of CL 10 d after mating, and in pregnant ewes, numbers of embryos 25 d after mating and lambs born, did not differ with treatment. In summary, low P₄ increased the size of the largest follicles and the age of the oldest ovulatory follicles. Embryos resulting from the ovulation of older and younger follicles in the same ewe did not differ in their ability to survive.     

Effects of nutrient restriction and melatonin supplementation on maternal and foetal hepatic and small intestinal energy utilization

To determine how nutrient restriction and melatonin supplementation influence ewe and foetal hepatic and small intestinal energy use, 32 primiparous ewes on d 50 of gestation were fed 60% (RES) or 100% (ADQ) of NRC recommendations with 0 (CON) or 5 mg/d (MEL) of dietary melatonin. On d 130 of gestation, small intestine and liver were weighed and collected. Data were analysed as a completely randomized design with a 2 × 2 factorial arrangement of treatments. Liver weight (g/kg EBW) decreased (p = 0.02) in RES ewes. Jejunum weight (g/kg BW) increased (interaction p = 0.04) in ADQ‐MEL ewes compared with all other treatments. Total in vitro O₂ consumption (mol/min/tissue) and total citrate synthase activity (mol/min/tissue and mol/min/kg EBW) in liver decreased (p ≤ 0.03) in RES ewes. Oxygen consumption (mol/min/kg EBW) increased (interaction p = 0.02) in jejunum of ADQ‐CON versus RES‐MEL and ADQ‐CON. Citrate synthase activity (mol/min/kg of EBW) increased (interaction p = 0.03) in jejunum of ADQ‐MEL compared with RES‐MEL and ADQ‐CON. Foetal liver weight (g/kg BW) decreased (p = 0.02) in RES versus ADQ. Foetal small intestine weight (g/kg BW) decreased (interaction p = 0.05) in RES‐MEL versus ADQ‐MEL. Total O₂ consumption (mol/min/tissue) and total citrate synthase activity (mol/min/kg of BW) in foetal liver decreased (p ≤ 0.05) in RES versus ADQ. Foetal small intestinal O₂ consumption (mol/min/kg of BW) was greater (interaction p = 0.03) in RES‐CON and ADQ‐MEL than RES‐MEL and ADQ‐CON. Maternal nutrient restriction had a greater effect than melatonin supplementation on liver and jejunum mass and energy utilization in dams and foetuses. Because intestinal mass and energy utilization were more responsive to melatonin supplementation in ewes fed adequate nutrition compared with restricted ewes, melatonin may have limited use as a therapeutic supplement to help overcome potential negative effects of nutrient restriction.     

Periconceptional undernutrition increases quantity and quality of oocyte population,but not cognitive or emotional response of 60‐day‐old lambs

Maternal periconceptional undernutrition is associated with altered development and increased risks of adverse outcomes in the offspring. The aim of this work was to determine the effect of periconceptional undernutrition on behavioural and reproductive aspects of the offspring. Fifty ewes were synchronized in oestrus (day 0) and allocated to two groups (n = 25) to be fed diets that provided 1.5 (C) or 0.5 (L) times the requirements for maintenance until day 15. Ewes were mated and fed the control diet from day 16 until lambing. Two months after lambing, 26 lambs were exposed to tests to determine their cognitive/emotional responses. Six ewe lambs were euthanized and in vitro oocyte maturation and fertilization procedures performed. The experimental diets produced no changes of mean live weight (LW) of C ewes, L ewes presenting a reduction in their initial LW with significant differences at day 15, in comparison with C ewes (p < 0.05). L ewes experienced a significant reduction in their body condition (BC) in comparison with C ewes (p < 0.05). Fourteen days after the onset of the experimental diets, mean LW and BC of L ewes was significantly lower than those of C ewes (p < 0.05). Undernourished ewes presented a trend to a reduction of prolificacy and fecundity (p < 0.10) in comparison with C ewes. Emotional and cognitive test revealed a similar response between groups. Ewe lambs from the undernourished ewes presented a population of oocytes 1.7 times higher than ovaries from control ewe lambs (66.0 ± 0.73 vs. 113.7 ± 15.6 oocytes; p < 0.05) and had more oocytes in the ‘good’ (p < 0.05) and ‘healthy’ (p < 0.05) categories. In conclusion, a low plane of nutrition around conception significantly increases quantity and quality of the oocyte population of 60‐day‐old female descendants. Modifications of the cognitive and emotional responses of the progeny have not been evidenced.     

Effects of season and superovulatory treatment on embryo yields in fine-wool Merinos maintained under field conditions

The aim of the study was to assess the effects of superovulatory treatment (multiple FSH‐dose vs single‐shot FSH treatment) and seasonality on embryo yields in fine‐wool Merino ewes. Treatment based on multiple FSH‐dose consisted of 200 mg of FSH (Folltropin^®) administered in seven decreasing doses. Single‐shot treatment consisted of a single dose of 70 mg of FSH + eCG. In ewes treated with multiple FSH doses, number of recovered embryos was higher (6.0 ± 0.5 vs 3.5 ± 1.0), while non‐fertilization rate was lower (12.8 ± 3.9 vs 40.3 ± 9.5) during the breeding season when compared to the non‐breeding season (p < 0.05); although similar values of recovered Grades 1–2 embryos were observed between seasons. During the breeding season, proportion of responding ewes (98.1 vs 57.1%), ovulation rate (13.9 ± 0.8 vs 3.2 ± 1.2), recovered structures (7.9 ± 0.6 vs 1.7 ± 0.7), total recovered embryos (6.0 ± 0.5 vs 1.2 ± 0.6) and good‐quality embryos (5.1 ± 0.5 vs 0.9 ± 0.6) were higher for the multiple FSH‐dose treatment than for the single‐shot protocol. In a similar way, in the non‐breeding season, ovulation rate (11.3 ± 1.8 vs 6.0 ± 1.1) and recovered structures (6.6 ± 1.2 vs 2.7 ± 0.6) were higher for the multiple FSH injections protocol than those for the single‐shot treatment, resulting in higher recovered Grades 1–2 embryos (3.2 ± 0.9 vs 1.4 ± 0.5). Current results indicate that seasonal anestrus affected embryo yields when applying multiple FSH‐dose superovulatory treatment in Merino ewes, by decreasing the number of recovered embryos although the number of recovered good‐quality embryos was not affected. During both seasons, multiple FSH injections produced higher ovarian response and number of viable embryos than the single‐shot treatment.     

Effects of plane of nutrition on in vitro fertilization and early embryonic development in sheep

Nutrition has been shown to influence several reproductive functions, including hormone production, oocyte competence and fertilization, and early embryonic development. To determine the effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 18; 47.0 +/- 1.5 kg of initial BW) were divided into control and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Pelleted diets containing 2.4 Mcal of ME/kg and 13% CP (DM basis) were fed once daily. During the first 4-wk acclimation phase, control and underfed ewes were fed 1,000 and 600 g/d, respectively. From wk 4 to 8, control (adequate) ewes were fed to maintain BW and offered 720 g/d, whereas underfed ewes received 432 g/d (60% restricted). Synchronization of estrus was performed using progestagen sponges for 14 d. Follicular development was induced by twice daily injections of FSH on d 13 (5 units/injection) and 14 (4 units/injection) of the estrous cycle. Oocytes were collected from all visible follicles on d 15 of the estrous cycle. After IVF, the proportion of developing embryos was evaluated throughout an 8-d culture period. Under-nutrition decreased (P < 0.006) the rate of cleavage, number of blastocysts per ewe, and rate of blastocyst formation (from 79 to 64%; from 3.3 to 0.8; and from 31 to 8%, respectively). However, the number of visible follicles, total number of oocytes, number of healthy oocytes, percentage of healthy oocytes, number of cleaved oocytes, and morula formation per ewe were similar for control and underfed ewes. These data indicate that undernutrition of donor ewes, resulting in lower BW and BCS, has a negative effect on oocyte quality, which results in lower rates of cleavage and blastocyst formation.     

Effects of Co‐culture of Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation on the Developmental Competence of Cloned Bovine Embryos

This study evaluated the effects of co‐culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co‐cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non‐co‐cultured somatic cell nuclear transfer (SCNT‐DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT‐DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation‐related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT‐DO group. Similarly, while the mRNA levels of the deacetylation‐related genes HDAC2 and HDAC3 were significantly higher in the SCNT‐DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co‐culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.     

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.     

Effects of melatonin on oocyte maturation in PCOS mouse model

The purpose of oocyte in vitro maturation is generation of mature oocytes that could support future development. Efforts have been made to enhance oocyte developmental competence by developing optimal culture conditions. The present study is conducted to determine melatonin effects on quality of polycystic ovarian syndrome (PCOS) oocytes when it has been added during in vitro maturation, and immature oocytes were cultured in defined conditioned medium with and without different melatonin concentrations. Melatonin could significantly improve nuclear maturation of PCOS oocytes (81.1% vs. 56.3%, P < 0.05 were achieved with 10^?6 mol/L concentration). Cleavage rate was significantly higher in 10^?5 mol/L concentration compared to untreated oocytes in PCOS (54% vs. 35%, respectively) and it was significantly higher with 10^?6 mol/L concentration in the control group, 55% versus 38%, compared to untreated oocytes. This study showed that melatonin has the potential to induce oocyte nuclear maturation and guarantee fertilization potential. © 2016 Japanese Society of Animal Science     

In vitro Maturation of Oocytes from Santa Ines Ewes Subjected to Consecutive Sessions of Follicular Aspiration by Laparoscopy

The success of embryo production in vitro depends upon the use of an efficient oocyte retrieval technique, and the best results have been obtained by laparoscopic aspiration. The aim of this study was to evaluate the effect of consecutive sessions of follicular aspiration on the quantity, quality and in vitro maturation competence of oocytes obtained from ewes subjected to hormonal stimulation. Six Santa Ines ewes underwent nine sessions of follicular aspiration by laparoscopy with a 7‐day interval between sessions, totalling 56 aspirations. After 24 h of culture, oocytes were stained and classified according to the stage of nuclear and cytoplasmic maturation. Oocyte retrieval rate was 61.4 ± 2%, resulting in a total of 249 oocytes. No significant variation was observed between sessions (p > 0.05). The average number of oocytes retrieved from each ewe was 6.4 ± 2 per session and 42 ± 4 in total. No significant difference was observed between the frequencies of the different stages of nuclear maturation: 32.72% mature, 40.74% immature and 26.54% degenerated/indeterminate oocytes; however, a significant difference was observed between the frequencies of the different stages of cytoplasmic maturation: 10.7% mature, 73.25% immature and 16.05% degenerated/indeterminate oocytes. No significant difference was observed in nuclear or cytoplasmic maturation between the weeks of procedure. We conclude that after nine consecutive sessions of follicular aspiration, the quantity and quality of retrieved oocytes remained unchanged as well as the levels of nuclear and cytoplasmic maturation obtained, demonstrating the viability of this technique for repetitive follicular aspirations on the same donor.     

Medium without ammonium accumulation supports the developmental competence of human embryos

L-Glutamine has been shown to play an important role during in vitro culture of mammalian embryos. However, it is easily decomposed into ammonium, which is believed to have deleterious effects on preimplantation embryos. In this study, we assessed prospectively the developmental competence of human embryos cultured in medium containing L-glutamine or a novel stable glutamine derivative and vitamins. The subjects of this study were 41 women who underwent IVF/ET treatment from September to November 2006 and from whom 6 or more oocytes were retrieved. Sibling oocytes were randomly divided into EA/BA (EmbryoAssyst and BlastAssyst containing a novel stable glutamine derivative and vitamins), and BAS groups (BlastAssyst system containing L-glutamine). There was no difference in pronuclear formation rate between EA/BA and BAS (74 vs. 69%). The blastulation rates of embryos based on the number of zygotes cultured in EA/BA on Days 5 (Day 0=insemination, 54%) and 6 (63%) were significantly higher (P<0.05) than those cultured in BAS (Day 5: 33% and Day 6: 45%, respectively). The present data indicate that a medium containing a novel stable glutamine derivative and vitamins supports the developmental competence of human embryos.     

Morphological Quality of Oocytes and Blood Plasma Metabolites in Repeat Breeding and Early Lactation Dairy Cows

The objectives of the study were to evaluate the morphological quality of oocytes in repeat breeder and early lactation cows and to determine the possible associations between the quality of oocytes and a range of blood metabolites. Oocyte quality and a range of metabolites were compared between 29 repeat breeder and 13 early lactation cows. The yield of oocytes from the repeat breeders was lower than that from the early lactation cows (4.4 ± 0.2 vs 5.4 ± 0.6, p < 0.05). Percentages of abnormal oocytes for the repeat breeders and the early lactation cows were 52.5% and 37.9%, respectively (p < 0.001). An excess of abnormal oocytes to normal was found in 55.2% of the studied repeat breeders (65.8% vs 34.2%, p < 0.05). Total protein, glucose and aspartate aminotransferase did not differ (p > 0.05) between the repeat breeders with an excess of abnormal oocytes (81 ± 1.0 g/l, 3.5 ± 1.0 mmol/l and 68.5 ± 3.7 U/l), those with the prevalence of normal oocytes (84 ± 1.0 g/l, 3.6 ± 0.1 mmol/l and 73.2 ± 3.5 U/l) and the early lactation cows (83 ± 2.0 g/l, 3.7 ± 0.1 mmol/l and 74.5 ± 3.6 U/I). The repeat breeders with an excess of abnormal oocytes had higher (p < 0.05) urea (5.2 ± 0.2 mmol/l) level than in those with the prevalence of normal oocytes (4.8 ± 0.2 mmol/l) and the early lactation cows (4.7 ± 0.2 mmol/l). A trend for higher total cholesterol and lactate dehydrogenase activity was found in the repeat breeders with an excess of abnormal oocytes. In conclusion, it is suggested that possible causes of repeat breeding in dairy cows may include impaired oocytes. An excess of abnormal oocytes in the repeat breeder cows was associated with elevated blood plasma levels of urea.     

Developmental Capacity In Vitro of Prepubertal Oocytes

A 2-year comparative study was carried out to evaluate the effect of ovary size, follicle size and oocyte quality of 3-month-old Simmental calves and the efficiency of using calf ovaries in an in vitro fertilization (IVF) programme. We evaluated the effects of different concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta (E-17beta) in the maturation medium on the in vitro development of calf oocytes into morula and blastocysts. The proportion of recovered oocytes (62.1%; 42.8%; 25.3%) and the percentage of good quality cumulus oocyte complexes (84.2%: 59.8%; 45.9%) decreased significantly (P < 0.01) with decreasing ovary size (L, M and S). The rates of two or more cells on Day 2 and of blastocysts on Day 7 and Day 9 were significantly lower (P < 0.01) for calf oocytes (61.5%; 18.9%: 15.9%) compared with those from sexually matured females (70.1%: 32.3%; 22.2%). Calf oocytes. matured in medium supplemented with 20 microg/ml or 10 microg/ml FSH plus 2 microg/ml E-17beta had higher rates of cleavage on Day 2 (64.1% and 64.7%) and blastocysts on Day 7 (24.5% and 22.4%) than the control supplemented with 10 microg/ml FSH (55.6% and 19.2%, respectively). Groups supplemented with 20 microg/ml FSH plus 2 microg/ml E-17beta and 10 mg/ml plus 4 mg/ml E-17beta showed a significantly lower developmental rate of blastocysts on Day 7 (14.6% and 14.5%). High concentrations of E-17beta (4 microg/ml) resulted in a significantly lower development of blastocysts on Day 9 (8.1%) and hatched blastocysts on Day 13 (3.5%) (P < 0.01). We conclude that the proportion of calf oocytes obtained from immature animals and their suitability for IVF are lower than those of cows. Thus, the use of oocytcs from sexually immature females would decrease the relative efficiency of IVF programmes. Supplementation with high concentrations of FSH can improve the maturation and developmental capacity of oocytes from prepubertal calves.     

High‐milking sheep have a lower ovulation rate and tend to yield fewer embryos in response to superovulation and intrauterine artificial insemination

Antagonistic relationship between milk yield and reproduction is reported in several livestock species. This study aimed to investigate whether genetic merit for milk production in dairy sheep affects responses to superovulation, embryo yield and quality. A total of 21 cross‐bred Sarda x Lacaune ewes homogeneous for age, parity and stage of lactation were included. The ewes were stratified as high‐producing or low‐producing based on their genetic merit for milk production estimated by a pentatrait repeatability animal model. Oestrus was synchronized using an intravaginal progesterone pessary inserted on Day 0 and removed on Day 14. Superovulatory treatment consisted of 350 I.U. of porcine FSH administered in eight decreasing intramuscular doses every 12 hr with a total dose of 10 ml of solution starting 12 days after insertion of sponges. Laparoscopic artificial insemination (AI) was performed 48 hr after pessary removal. Surgical embryo recovery was performed at Day 8 after pessary removal. Correlation between breeding value for milk production and the number of corpora lutea (CL) was significantly different from zero (?0.49). High‐producing ewes had a lower number of CL than low‐producing counterparts (7.6 ± 2.50 vs 12.1 ± 5.16 respectively; p < .02). Furthermore, there was a tendency for high‐producing ewes to yield fewer embryos than low‐producing females (5.3 ± 3.46 vs 9.18 ± 5.11; p = .09). No differences were observed between ewes in both genetic groups with regard to the number of embryos of grades 1, 2 and 3. To our knowledge, this is the first report highlighting an antagonism between genetic merit for milk production and the ability to produce embryos in sheep. These results deserve to be considered in sheep breeding programmes.     

Reestablishment of transzonal projections and growth of bovine oocytes in vitro

Transzonal projections (TZPs) that maintain bidirectional communication between oocytes and granulosa cells or cumulus cells are important structures for oocyte growth. However, whether TZPs develop between TZP-free oocytes and granulosa cells, and whether reestablished TZPs support oocyte growth, is unknown. We first examined changes in TZPs after denudation of bovine oocytes collected from early antral follicles (0.5–0.7 mm). Twenty-four hours after denudation, almost all the TZPs disappeared. We also examined the reestablishment of TZPs by coculturing TZP-free denuded oocytes (DOs) with mural granulosa cells (MGCs) collected from early antral follicles. In addition, to confirm if the reestablished TZPs were functional, the reconstructed complexes (DO+MGCs) were subjected to in vitro growth culture and found that the MGCs adhered to TZP-free DOs and TZPs were reestablished. During in vitro growth culture, DO+MGCs developed and formed antrum-like structures. After culture, the number of TZPs in DO+MGCs increased, and the oocytes grew fully and acquired meiotic competence. These results suggest that reestablished TZPs are able to support oocyte growth.