Dietary omega-3 fatty acids from linseed oil improve quality of post-thaw but not fresh sperm in Holstein bulls.

Docosahexaenoic acid (DHA), a member of the n-3 fatty acid family present in fish oil, has several positive effects on bovine sperm, including membrane integrity, motility and viability, as well as cold sensitivity. Our objective was to investigate effects of varying amounts of omega-3 fatty acids from linseed oil, administered orally, on quality of fresh and frozen-thawed bull sperm. Twenty fertile Holstein bulls (874 ± 45.38 kg) were randomly and equally assigned to four groups and received encapsulated (rumen-protected) fats for 12 weeks, as follows: group P, 300 g palm oil; group Pl, 200 g palm oil + 100 g linseed oil; group pL, 100 g palm oil + 200 g linseed oil; and group L, 300 g linseed oil. Sperm quality of fresh and frozen-thawed semen was evaluated by routine assays including sperm motion characteristics (CASA), membrane integrity (eosin-nigrosin), membrane activity (hypo-osmotic swelling test; HOST) and malondialdehyde (MDA) content. There were no significant differences among groups in semen volume, sperm concentration or sperm quality parameters in fresh semen. However, after freezing-thawing, total and progressive motility in group P (59.61 ± 1.95 and 40.19 ± 2.48%, respectively; LSM ± SEM) were lower (P < 0.05) than in groups Pl (66.06 ± 1.95 and 47.53 ± 2.48%), pL (65.67 ± 1.95 and 47.48 ± 2.48%) and L (65.36 ± 1.95 and 47.62 ± 2.48)%, with no significant differences among the latter three groups. Furthermore, membrane integrity (eosin-nigrosin) and activity (HOST) were lower (P < 0.05) in group P (55.79 ± 2.15 and 42.19 ± 2.17%) compared to groups Pl (62.73 ± 2.15 and 48.93 ± 2.17%), pL (64.06 ± 2.15 and 50.01 ± 2.17%) and L (64.47 ± 2.15 and 49.68 ± 2.17%), with no significant differences among the latter three. Furthermore, there were more (P < 0.05) morphologically abnormal sperm in group P (25.99 ± 1.62%) than in groups Pl, PL and L (21.55 ± 1.62, 21.69 ± 1.62 and 20.90 ± 1.62%). In conclusion, feeding Holstein bulls 100-300 g linseed oil daily improved sperm cryotolerance.

Determination of optimum carcass weight for meat quality and fatty acid composition in fat-tailed male and female Chall lambs.

The aim of this study was to determine the optimum carcass weight for meat quality and fatty acid composition in fat-tailed Chall lambs. Thirty lambs (15 male and 15 female) were allotted to three carcass weight groups: (1) light carcass weight (LCW 10-15 kg), (2) moderate carcass weight (MCW 15-20 kg), and (3) heavy carcass weight (HCW 20-25 kg). Back fat thickness and intramuscular fat (IMF) content were greater (P < 0.05) for HCW and female groups than their counterparts, respectively. Drip loss was lower (P < 0.05) for female and HCW lamb groups than male and LCW group, respectively. Female and LCW lambs had lower (P < 0.05) shear force compared with their corresponding male and HCW groups. Meat from LCW and MCW lambs had higher lightness (L* value; 43.6, 43.5 vs. 39.9), while redness (a* value; 13.6, 13.9 vs. 15.4) was greater for HCW and female (13.7 vs. 14.9) lambs compared with their counterparts (P < 0.05). The MCW lambs produced meat with higher overall acceptability compared with other two groups (P < 0.05). The HCW lambs contained lower polyunsaturated fatty acids (PUFA), polyunsaturated fatty acids to saturated fatty acids (P:S) ratio, and n-3 PUFA compared with LCW group (P < 0.05). Results show that as the animal grow faster and achieved HCW, the IMF content also increased mainly as storage triglyceride, while functional fats consisting long-chain omega-3 did not increase proportionately. In addition, the study also demonstrates that using IMF for predicting or assessing meat quality aspects such as juiciness and flavor or the nutritional value of meat relating to health claimable fatty acids would not be appropriate.

MitoQ preserves the quality and fertility of liquid-preserved ram sperm.

Supplementing the semen extender with some antioxidants may preserve sperm quality following liquid preservation. The aim of the current study was to evaluate the influence of the use of MitoQ in the semen extender on quality parameters and fertility of liquid-preserved ram semen. In this study, diluted semen samples were divided into five parts and supplemented with 0, 1, 10, 100 and 1000 nM MitoQ. The prepared samples were stored at 3-5 °C for up to 50 h. Motility, viability, mitochondrial activity, membrane integrity, and malondialdehyde concentration of the chilled sperm were assessed at 0, 25, and 50 h. To evaluate reproductive performance, artificial insemination was performed with semen liquid-preserved for 25 h. In results, at 0 h, no difference between the groups was observed. The use of 10 and 100 nM MitoQ resulted in higher (P ≤ 0.05) total motility, progressive motility, membrane integrity, mitochondrial activity, viability, and lower malondialdehyde concentration than the other groups after 25- and 50-h storage. Pregnancy, parturition and lambing rates were higher (P ≤ 0.05) when ewes were inseminated with 25-h chilled semen samples containing 10 and 100 nM MitoQ compared to the control. Therefore, supplementing the semen extender with MitoQ (10 and 100 nM) could be an efficient method to improve the quality and fertility rate of liquid-preserved ram semen.

Effect of different surgical methods in the mouse embryo transfer: Electrosurgery versus cold surgical technique effects on repeated use of surrogate mothers, pregnancy rate and post-surgical behavior.

Using the cold surgical technique (CST) is the most common practice to accomplish embryo transfer (ET). However, it can lead to uncontrolled bleeding and mortality in laboratory animals. Electrosurgery technique (EST) has provided the opportunity to prevent such complications. This study was aimed to evaluate CST versus EST in terms of repeated use of surrogate mothers, litter size, implantation rate and post-surgical behavior. Virgin female NMRI mice were allocated into two different surgical groups (n = 40): 1) CST-ET (control) and 2) EST-ET. Results showed that the first ET in EST-ET and CST-ET groups did not affect litter size, pregnancy rate and survival of surrogate mothers. Following the second and the third ETs, litter size was significantly affected through CST compared to EST, pregnancy rate and survival of surrogate mothers. Litter size, pregnancy rate and surrogate mothers survival rate did not show any significant reduction following the first and the second ETs in EST group. On the other hand, the third ET showed dramatic reduction for all aforementioned parameters regardless of the chosen surgical method for ET. Mice in EST-ET group did not show any significant change in their behavior indicating reduced well-being during the first 24 hr following the first, the second and the third ETs compared to CST-ET group. In conclusion, using EST for ET in mouse made it feasible to reuse surrogate mothers with minimum animal mortality; this could be pivotal with regard to reproductive and animal welfare aspects and research costs. Also, the results indicated that bleeding has severe diverse effects on ET efficiency.

Effects of co-incubation with conspecific ampulla oviductal epithelial cells and media composition on cryotolerance and developmental competence of in vitro matured sheep oocytes.

Developmental potential of cryopreserved in vitro matured oocytes is very low in nearly all mammalian species studied to date. Despite relatively high cleavage rates, the vitrified/warmed metaphase II oocytes have a decreased rate of blastocyst formation, which can be attributed to the elevated cytoplasmic lipid content and lipid droplet fragmentation. Secretory products of ampulla oviductal epithelial cells (AECs) at the periovulatory stage of the ovarian cycle enhance the viability of in vitro matured oocytes. The present study was undertaken to determine if co-culture of cumulus-oophorus complexes (COCs) with conspecific AECs or reducing the lipid content of in vitro matured ovine oocytes would improve their cryotolerance and ensuing developmental competence. Ovine COCs aspirated from the slaughterhouse ovaries were matured in the following media or culture conditions: TCM199 + FBS + AECs (T1); TCM199 + FBS (T2); TCM199 + BSA (T3); TCM199 + 0.6 mg/mL of l-carnitine (T4); TCM199+ l-carnitine + FBS (T5), or TCM199 only (Ctr). Subsequently, the oocytes were vitrified and used for in vitro fertilization (IVF). The lowest degree of zona pellucida (ZP) hardening following vitrification of in vitro matured sheep oocytes was observed in T1 and T5 (P < 0.05). Cleavage, blastocyst formation and ensuing development (i.e., total cell numbers) as well as blastocyst hatching rates were all greater (P < 0.05) in T1 compared with the remaining groups; in vitro matured COCs in T4 and Ctr did not develop beyond the cleavage stage. The inner cell mass: trophectoderm cell ratio in T1 (1:3.29) was significantly greater compared with T2 (1:3.39), T3 (1:3.40) and T5 (1:3.44). The present results indicate that the ovine COCs/AECs co-culture system had the most positive influence on cryotolerance, ZP hardening, and developmental competence of in vitro matured oocytes.

The Effect of Oral Administration of Zinc Oxide Nanoparticles on Quantitative and Qualitative Properties of Arabic Ram Sperm and Some Antioxidant Parameters of Seminal Plasma in the Non-Breeding Season.

Zinc is an essential mineral and accepted as a trace element in the animal nutrition and for its role in biological enzymatic pathways. This study aimed to investigate the effect of oral administration of zinc oxide nanoparticles (ZnONPs) on some of the antioxidant parameters of semen plasma, quantitative and qualitative properties of Arabic ram sperm in the non-breeding season. Twelve adult Arabic rams (about 3-5 years old, 70 ±2.1 kg) were randomly assigned to receive one of the three levels of dietary ZnONPs (control; 0, group 1; 40 ppm and group 2; 80 ppm). Results showed that using different levels of ZnONPs increased the activity of superoxide dismutase enzyme (SOD) and total antioxidant capacity (TAC) of semen plasma significantly compared with the control group (P<0.05). Motility (74.83%), viability (76.90%), semen volume (1.76 ml) and sperm concentration (1418×106/ml) were significantly (P<0.05) higher in ZnONPs supplemented groups compared with the control group. The sperm morphological abnormalities reduced significantly in treated groups (10.46 and 9.07%) compared with the control group (12.66%; P <0.05). Also, the results suggested that 80 ppm level of ZnONPs increased the functionality of sperm membrane (44.38%) compared with other groups (37 and 35.66%, respectively for groups 1 and control) (P<0.05). Based on the results, using 80 ppm level of ZnONPs lead to an improvement in the activity of superoxide dismutase enzyme (48.62 ml) and total antioxidant capacity of semen plasma (111.88 µg/ml) compared with other groups (P<0.05). In conclusion using 80 ppm level of ZnONPs had a positive effect on the quantitative and qualitative properties of sperm and lead to a significant betterment in the activity of some antioxidant parameters of Arabic ram semen in the non-breeding season.

Effect of melatonin supplementation in the long-term preservation of the sheep ovaries at different temperatures and subsequent in vitro embryo production.

Investigations in the past decades have shown that oocytes developmental competence following in vitro fertilization is greatly influenced by an interval between isolation of the ovaries immediately after death/slaughter and oocytes recovery from the visible follicles. In order to determine the optimal conditions for long-term preservation of ovaries, an experiment was conducted with adding different doses of melatonin (0 (C), 500 (M1), 600 (M2), 700 (M3) and 800 (M4) µM) as an antioxidant to sheep ovaries preservation medium (PBS) maintained at 4 and 20 °C for 24 h. The effects on in vitro embryo production (IVEP) parameters including maturation, fertilization, cleavage, and blastocyst rates and the total number of blastomere were evaluated after the ovaries preservation. Melatonin reduced the decline in fertilization rate as an indicator of success in vitro maturation (P ≤ 0.05). Furthermore, ovarian storage time had significant negative effect (P ≤ 0.05) on IVEP parameters. Supplementation with melatonin increased the total cell number of blastocysts as an indicator of embryo quality (i.e. mean blastomeric cells in 4°C groups: 86.00 ± 3.00, 98.50 ± 3.5, 111.5 ± 1.5, 125.5 ± 2.00 and 126.50 ± 5.5 for C, M1, M2, M3 and M4. respectively). Overall, the results showed that the use of melatonin antioxidant in the ovaries storage medium had beneficial effects on sheep oocytes development and embryos quality.

The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep.

The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P < 0.05) for oocytes obtained from ewes (G1, G4, and C1) compared with those from ewe lambs (G2, G3, and C2). The greatest increment in MPF activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM = 3.62) followed by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89, G1: 1.87, and G4: 1.86). In summary, coincubation with AAE during the 24-hour IVM had a positive impact on ovine oocyte competence and ensuing in vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the improved rate of blastocyst formation after IVF of prepubertal sheep oocytes.

Differential influence of ampullary and isthmic derived epithelial cells on zona pellucida hardening and in vitro fertilization in ovine.

The central role of the oviduct, as the site of zona pellucida (ZP) maturation, fertilization and early embryogenesis, has been recognized. The objective of this study was to investigate whether ampullary and isthmic derived epithelial cells have different effects on in vitro ZP hardening, in vitro fertilization (IVF) and in vitro culture (IVC) of the resulting embryos. Cumulus oocyte complexes (COCs) were matured in a coculture system with ampullary/isthmic epithelial cells, TCM199 supplemented with insulin-like growth factor I (IGF-I) and epithelial derived growth factor (EGF) (GF treated group), conditioned media produced using ampullary (ACM), isthmic (ICM), COCs+ampullary, and COCs+isthmic epithelial cells, contactless culture system, oviductal fluid, GF+ACM/ICM, and drops of TCM199 (control), for 24h. The matured oocytes were randomly divided into two groups: Group I was subjected to ZP digestion; Group II underwent IVF. The duration of the ZP digestion, in a coculture system with ampullary epithelial cells (AE) was significantly increased (p<0.05), compared with other groups. Penetrated oocytes and monospermic fertilization were significantly increased (p<0.05) in the AE group. The mean number of spermatozoa per penetrated oocyte was reduced dramatically for the AE group (p<0.05). A significant increase (p<0.05) in the embryo development was observed in all treated groups, compared to the control. Results revealed that epithelial cells harvested from the ampullary segment of the oviduct had in vitro specialized role in ZP hardening and have subsequent IVF and IVC outcomes.

Dietary n-3 PUFAs improve fresh and post-thaw semen quality in Holstein bulls via alteration of sperm fatty acid composition.

The goal of this study was to investigate the effect of fish oil-supplemented diet on fresh and post-thaw semen quality and sperm lipid composition in bulls. Bulls were randomly assigned to two groups (n = 6). Six bulls were used as the control group and six received the fish oil (1.2% dry matter of total diet) for 11 weeks. Semen was individually collected from each bull and frozen biweekly. Semen volume, sperm concentration, viability, progressive motility, and fatty acid profile of sperm were measured in 1st, 3rd, 5th, 7th, 9th, and 11th week of experiment. Viability, progressive motility, and fatty acid profile of post-thaw sperm were also measured in 3rd, 5th, 9th, and 11th week of experiment. Data were analyzed with using Proc GLM or MIXED (for repeated measurement data) in SAS program. The fish oil-supplemented diet increased the semen volume and sperm concentration. The fish oil-supplemented diet also altered the viability, progressive motility, and fatty acid profile of fresh and post-thaw sperm. In conclusion, feeding a fish oil-enriched diet via alteration of fatty acid profile of sperm lipid could improve in vitro quality of fresh and post-thaw sperm in Holstein bulls.

In vitro ovine embryo production: the study of seasonal and oocyte recovery method effects.

BACKGROUND: To current knowledge, different oocyte's recovery method and various seasons have profound impact on in vitro embryo production (IVEP). OBJECTIVES: The aim of this study was to define an efficient recovery method for oocytes harvesting from slaughterhouse material in different seasons, and their effects on IVEP yield. MATERIALS AND METHODS: Ovaries from slaughtered ewes in breeding season (BS) and non-breeding season (NBS) were collected from a local abattoir. The oocytes were recovered through aspiration, centrifugation (ORC), puncture and slicing, and categorized into three classes (I, oocytes with more than three layers of cumulus cells; II, less than three layers with damaged cumulus cells; III, denuded oocytes). After cultivation in TCM 199 for 24 hours, matured oocytes were subjected to in vitro fertilization (IVF) and in vitro culture (IVC). The oocyte recovery using ORC in BS and NBS was significantly higher (P < 0.05) compared with other recovery methods. RESULTS: No significant dissimilarities in the proportion of oocytes reaching M-II stage were recorded when using different oocyte recovery methods in different seasons. Aspiration resulted in lower (P < 0.05) proportion of class I (BS, 60.0 ± 2.1; NBS, 51.1 ± 2.1) compared to ORC (BS, 82.0 ± 1.2; NBS, 70.0 ± 1.2), slicing (BS, 80.0 ± 2.1; NBS, 71.0 ± 1.4) and puncture (BS, 80.0 ± 1.5; NBS, 72.0 ± 2.0). Monospermy and blastocyst development rates were significantly higher using ORC than other recovery techniques in both BS and NBS. More oocytes with high quality, greater blastocyst development and oocyte recovery rates were achieved in BS. CONCLUSIONS: The results revealed that oocytes harvesting technique and season are effective in the rate of cleavage and blastocysts' development, and suggest that despite same meiotic resumption rate in all treatments, it would be better to use ORC.