Addition of MitoTEMPO to the maturation medium improves in vitro maturation competence of bovine oocytes.

The effects of MitoTEMPO, a mitochondria-targeted antioxidant, and its non-targeted parent, TEMPO, on bovine oocyte maturation competence have not been determined so far. Hence, our study was aimed to investigate the effects of supplementing maturation medium with different concentrations of MitoTEMPO (0.00, 0.10, 1.00 and 10.00 µM) or TEMPO (0.00, 5.00, 10.00 and 15.00 mM) on in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. The oocytes after IVM and IVF were evaluated for the signs of nuclear maturation and normal fertilization. The average number of spermatozoa penetrated per oocyte and the level of intracellular reactive oxygen species (ROS) were also evaluated. The results showed that percentages of bovine oocytes reached the metaphase II stage of meiosis were significantly higher in the 1.00 µM MitoTEMPO group compared to the control group (without antioxidant supplementation). The normal fertilization rate also tended to be greater in this group than the control group. In comparison with the control group, the medium supplementation with 1.00 µM MitoTEMPO led to a significant decrease in the intracellular ROS level. The average number of spermatozoa penetrated per oocyte was not significantly different among the antioxidant-treated and the non-treated groups. The TEMPO addition to the maturation medium affected neither the rate of maturation/fertilization nor the level of intracellular ROS in bovine oocytes. Based on these results, we concluded that MitoTEMPO at a concentration of 1.00 µM had beneficial effects on the quality and fertilization potential of bovine oocytes.

Influence of seasonal differences on semen quality and subsequent embryo development of Belgian Blue bulls.

Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14-28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of the two groups was determined in vitro. Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to the high THI group (P ≤ 0.05), whereas semen volume, subjective motility, and total sperm output were not affected (P > 0.05). In frozen-thawed semen, total and progressive motility, viability, and straight-line velocity were lower in high THI compared to the low THI group (P < 0.05), while H2O2 concentration, aberrant chromatin condensation, and abnormal spermatozoa were higher in the high THI group (P < 0.05). Blastocyst rates were significantly higher when low THI samples were used (P < 0.05). Moreover, the total cell number and trophectoderm cells were significantly higher (P < 0.05) in blastocysts derived from low THI samples, whereas the apoptotic cell ratio was significantly higher (P < 0.01) in blastocysts derived from high THI spermatozoa. In summary, our data show that elevated ambient temperature and humidity during summer can decrease the quality of frozen-thawed spermatozoa in Belgian Blue bulls and also affect subsequent embryo development.

Influence of Different Combinations of Permeable and Nonpermeable Cryoprotectants on the Freezing Capacity of Equine Sperm.

This study was aimed to evaluate the effect of permeable cryoprotectants in combination with trehalose or sucrose on the freezing capacity of stallion sperm. For this purpose, the ejaculates (n = 24) were collected from four healthy mature Turkmen stallions. The ejaculates were pooled and diluted with one of the extenders containing a combination of 5% of permeating (dimethylacetamide [DMA]; dimethylformamide [DMF] or glycerol) and 50 mM of nonpermeating cryoprotectant agents (CPAs) (sucrose or trehalose) to a final concentration of 200 × 106 spermatozoa/mL. The extended samples were cryopreserved and thawed using a standard protocol. The samples were evaluated for motion kinetics, morphological abnormalities, plasma membrane functionality (PMF), viability, and lipid peroxidation. The results showed that the sperm cryopreserved in extender containing DMA produced higher (P ≤ .05) total motility, straightness, straight line velocity, curvilinear velocity, and lower (P ≤ .05) lipid peroxidation (malondialdehyde [MDA] concentration) compared with DMF and glycerol groups. Overall, both DMA and DMF have shown higher (P ≤ .05) sperm motion kinetics, viability, PMF, and lower (P ≤ .05) morphological abnormalities and MDA concentration compared with the glycerol. However, except morphological abnormalities, all of the other parameters did not differ between trehalose and sucrose. Likewise, there was no interaction between permeating and nonpermeating CPAs (P ≥ .05) except in terms of sperm abnormalities (P ≤ .05). In conclusion, the use of DMA or DMF as alternative CPAs of glycerol could be more effective for successful cryopreservation of stallion sperm. The nonsignificant interaction between permeating and nonpermeating CPAs for most of the post-thaw sperm parameters negates possible synergism among these compounds.

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.

Supplementation of extender with coenzyme Q10 improves the function and fertility potential of rooster spermatozoa after cryopreservation.

The effects of coenzyme Q10 (CoQ10) has not yet been assessed for cryopreservation of rooster semen. The aim of this study was to evaluate the effect of different concentrations of CoQ10 in Lake extender for cryopreservation of rooster semen. The viability and apoptosis status, DNA fragmentation, abnormal morphology, motion parameters, membrane functionality, mitochondrial activity, acrosome integrity, lipid peroxidation, and fertility potential were evaluated after the freeze-thaw process. Semen samples were collected from ten roosters, twice a week, and then diluted in extender contained different concentrations of CoQ10 as follows: Lake without CoQ10 (control, Q 0), Lake containing 1 µM (Q 1), 2 µM (Q 2), 5 µM (Q 5), and 10 µM (Q 10) CoQ10. Supplementation of Lake with 1 and 2 µM CoQ10 resulted in greater sperm viability, total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, and fertility rate. Furthermore, the extent of lipid peroxidation in thawed spermatozoa treated with 1 and 2 µM CoQ10 was less than with the other groups. Different concentrations of CoQ10 had no effect on DNA fragmentation and sperm morphology. Results of the present study indicate that supplementation of Lake extender with 1 and 2 µM CoQ10 enhances the quality of rooster sperm after the freeze-thaw process.

Effect of feeding pomegranate seed oil as a source of conjugated linolenic acid on Arabian stallion semen quality in cooled and postthawed condition.

The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen-thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty-day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation were analysed. In frozen-thawed semen, sperm dynamic characteristics were analysed by CASA, acrosome status and mitochondrial activity (evaluated by Flow cytometry) determined. The effects of treatment, time, semen type and their interactions were submitted to PROCMIX (SAS® ), and means compared by the Tukey test. Also, collected semen samples were artificially inseminated to evaluate fertility and pregnancy rate after day 60 of the experiment. The results from fresh condition showed that semen volume, sperm concentration, abnormality and live sperm were not affected by dietary treatment (p > 0.05). In cooled condition, the higher value for sperm plasma membrane integrity and viability was observed in PSO group compared to control after 24 hr cooled and stored in 5°C. In postthawed condition, the higher value for CASA total motility and acrosome status was observed in PSO group compared to control group (p < 0.05). One hundred and twenty-six mares were artificially inseminated for fertility trial using control and PSO groups' fresh semen. The average pregnancy rates were not significantly different between control and treated group (62.88% and 65.90%, respectively) (p > 0.05). We concluded that under the conditions of this study, dietary supplementation of 200 ml pomegranate seed oil seems to relatively improved Arabian horse sperm quality during storage in cooled and frozen condition via increasing plasma membrane integrity, viability and acrosome status, but did not improve the pregnancy rates.

D-Aspartate amends reproductive performance of aged roosters by changing gene expression and testicular histology.

Male broiler breeders (n=32) of 55 weeks of age were administered four different doses of capsulated d-aspartate (DA; 0, 100, 200 or 300mgkg-1day-1, p.o. (DA0, DA100, DA200 and DA300 respectively)) for 12 successive weeks to assess reproductive performance, blood testosterone, testicular histology and transcript levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), androgen receptor (AR), LH receptor (LHR), 3ß-hydroxysteroid dehydrogenase (3BHSD), proliferating cell nuclear antigen (PCNA), glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) and glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B). Blood samples and ejaculates were collected, and bodyweight was recorded weekly for 10 weeks. AI was performed weekly for the last 2 weeks to determine the number of sperm penetration holes in the perivitelline layer, fertility and hatchability. Testes histology and transcript levels were evaluated in the 12th week. Bodyweight, numbers of Leydig cells and blood vessels, testis index and levels of sperm abnormalities were not affected (P>0.05) by the treatment. However, sperm total and forward motility, plasma membrane integrity and functionality of sperm, ejaculate volume, testosterone concentration and fertility were higher (P<0.05) in both the DA200 and DA300 groups compared with the other groups. In the DA100 and DA200 groups, sperm concentration, number of spermatogonia, thickness of the seminiferous epithelium and the diameter of tubules were significantly higher (P<0.05) than the other DA-treated groups. The number of penetration holes, hatchability and malondialdehyde concentration were higher in the DA200, all DA-treated and DA300 groups respectively compared with the control and other treatment groups. Except for P450scc, AR, LHR and PCNA transcript levels in the DA300 groups, the relative expression of the genes evaluated improved significantly in the other DA-treated groups. Based on these experimental findings, it is concluded that DA improves reproductive performance of aged roosters.

Does MnTBAP ameliorate DNA fragmentation and in vivo fertility of frozen-thawed Arabian stallion sperm?

Overproduction of reactive oxygen species during sperm freeze-thawing process leads to membrane lipid peroxidation, DNA damage, motility loss, and subsequent death. This oxidative stress can be alleviated by the addition of some antioxidants to semen extenders prior to freezing. This study was performed to evaluate the in vitro effectiveness of MnTBAP (a cell permeable antioxidant) on stallion sperm freezability and in vivo fertility rate. Twenty-one ejaculates were, collected with missouri model artificial vagina (n = 3 stallions, seven ejaculate each), and diluted (1:2 v/v) with phosphocaseinate base INRA extender, containing 0 (control), 100, 200 and 300 µM of MnTBAP and frozen using acontrolled-rate freezing system. The following parameters were determined: sperm motility, viability, membrane integrity, acrosome abnormalities, lipid peroxidation and DNA fragmentation. MnTBAP improved horse semen quality parameters in a dose-dependent manner. The100 µM concentration of MnTBAP did not show a significant difference in semen parameters compare with control group (p > 0.05). Accordingly, the extender supplemented with 200 µM resulted in higher sperm total and progressive motility (55.3 ± 4.28% and 33.2 ± 2.90%), viability (43.9 ± 2.14%), and membrane integrity (50.8 ± 2.14%), provided a greater protective effect in the percentage of total abnormalities compare to other groups (p < 0.05), and showed lower sperm with damaged DNA with lower MDA levels (p < 0.001). Higher concentrations (300 µM) not only did not improve the results but inversely affected sperm parameters. Twelve mares were used for fertility trial in the cross over study of 60 deep horn inseminations performed using control (9/30 pregnancy/mare) and 200 µM - MnTBAP (14/30 pregnancy/mere) groups frozen semen. The Average pregnancy rates were not significantly different between control and treated group (30% and 46.66%respectively) (p > 0.05). Under the conditions of this study, 200 µM - MnTBAP reduced the oxidative stress and protect the DNA fragmentation of Arabian stallion sperm during cryopreservation; but did not improved pregnancy rates.

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.

Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen.

This study was performed to evaluate the effectiveness of quercetin as a non-enzymatic antioxidant in combination with glycerol or Dimethylacetamide (DMA), on freezability of goat semen. Ejaculates from four healthy mature Mahabadi goats were collected using an artificial vagina. After primary processing, semen was pooled and extended by egg yolk based extender supplemented with different concentrations of quercetin (10 or 20 µM) along with 5% glycerol or DMA. The extended semen was frozen and sperm motility parameters, viability, abnormality, membrane integrity and lipid peroxidation were assessed after thawing. Results showed that sperm viability, total motility, progressive motility, straightness (STR) and linearity (LIN) were higher (P < 0.05), and abnormality percentage and MDA concentration were lower (P < 0.05) in extender containing DMA. Similarly, higher (P < 0.05) total motility, progressive motility, viability and membrane integrity along with lower (P < 0.05) MDA level were noted in Q10 group. The lowest (P < 0.05) MDA level was observed in DMA extender containing moderate level of quercetin (Q10D). Also the STR was higher (P < 0.05) in Q10D compared to Q10G and Q20G groups. In conclusion, supplementation of extender with 10 µM quercetin in combination with DMA improves the goat sperm motion kinetics and suppresses lipid peroxidation after freezing and thawing. Furthermore, DMA is more effective cryoprotectant for the freezing of goat sperm.

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.

Improvement of post-thawed sperm quality and fertility of Arian rooster by oral administration of d-aspartic acid.

This study was conducted to investigate the effect of d-Aspartic acid (D-Asp) on post-thawed sperm quality, fertility and hatchability outcomes in male broiler breeders. Twenty 55-week-old roosters were selected and equally split into four groups (n = 5 rooster/group). Different daily D-Asp doses including 0 (D-0), 100 (D-100), 200 (D-200) or 300 (D-300) mg/kg BW were capsulated and individually administered for 12 weeks to roosters in each group. Semen samples were weekly collected from 7th to 12th week of experiment. Sperm quality from 7th to 11th week was evaluated in both fresh (total and forward motility and plasma membrane functionality) and post-thawed (total and forward motility, plasma membrane functionality, apoptosis status and mitochondrial activity) conditions. Also, collected semen samples on the 12th week were frozen and artificially inseminated to evaluate fertility and hatchability. The results from fresh condition showed that total and forward motility and plasma membrane functionality were significantly higher in D-200 compared to other groups. Also, interaction effect of time and treatment was not significant for all assessed parameters in fresh condition. In post-thawed condition, D-200 showed significantly higher total and forward motility, fertility and hatchability compared to other groups. The higher value for plasma membrane functionality and mitochondrial activity was observed in D-200 compared to D-0 and D300 groups. However, the percentage of live, early apoptotic and dead spermatozoa were not significantly affected by applied treatment in the current study. No significant difference for time and treat interaction effect was observed for all assessed parameters except forward motility. In conclusion, it seems that D-Asp administration could improve fresh and post-thawed sperm quality and post-thawed sperm fertility in male broiler breeders.

Growth performance parameters, bone calcification and immune response of in ovo injection of 25-hydroxycholecalciferol and vitamin K3 in male ross 308 broilers.

This experiment aimed to evaluate the effects of in ovo injection of 25-hydroxycholecalciferol (25-OH-D3) and Vitamin K3 on growth performance, bone calcification and immune system responses in male Ross 308 broilers. Twelve treatment groups with a total number of 768 experimental hatching eggs, four replications and 16 eggs in each replication were selected to form a completely randomized design of factorial arrangement. Treatments included: (1) distilled water, (2) 0.4 µg D3, (3) 0.4 µg D3 + 2 µg K3, (4) 0.4 µg D3 + 6 µg K3, (5) 0.6 µg D3, (6) 0.6 µg D3 + 2 µg K3, (7) 0.6 µg D3 + 6 µg K3, (8) 0.8 µg D3, (9) 0.8 µg D3 + 2 µg K3, (10) 0.8 µg D3 + 6 µg K3, (11) 2 µg K3 and (12) 6 µg K3. Eggs were transferred to corresponding hatching baskets on the 18th day of incubation and received 0.5 ml of experimental solutions specific to each treatment. The results of our experiments showed that Treatment No. 4 ranked the best out of those administered; holding the highest level of weight gain, feed intake during the breeding period (grower and finisher), bone calcium and phosphorus concentration, and tibia fractural force, (p < 0.05). Treatment No. 4 also showed a significant increase in antibody titer against the SRBC. Maximum stimulation to PHA injection also belonged to this treatment. In contrast, treatment No. 1 held the greatest alkaline phosphates amount (p < 0.05). No improvements were observed in calcium egg shells compared to the control group. Our data implies that appropriate levels of Vitamins D3 and K3 in ovo injection has beneficial effects on growth performance, immune system and bone development.

Fertility response of artificial insemination methods in sheep with fresh and frozen-thawed semen.

The aim of this study was to evaluate the fertility response of artificial insemination (AI) methods with fresh and frozen sperm in sheep. In experiment 1, one hundred and fifty fat tailed Zandi ewes were assigned into 3 equal groups and inseminated with three AI methods consisting of vaginal, laparoscopic and trans-cervical AI with fresh semen. In experiment 2, a factorial study (3 AI methods × 2 extenders) was used to analyze the effects of three AI methods and two freezing extenders containing soybean lecithin (SL) or Egg yolk (EY) on reproductive performance of 300 fat tailed Zandi ewes. Also, total motility, progressive motility, viability and lipid peroxidation of semen were evaluated after freeze-thawing in two extenders. In result, there was no significant difference among three AI methods when fresh semen was used. In experiment 2, the highest percentage of pregnancy rate, parturition rate and lambing rate were obtained in laparoscopic AI group (P < 0.05). Although pregnancy rate, parturition rate and lambing rate in trans-cervical group were higher (P < 0.05) than vaginal group, the results were not as high as laparoscopic group. No difference was observed between SL and EY extenders and their performance was close to each other. It can be concluded that although no difference was observed on reproductive performance for fresh semen, trans-cervical AI was more efficient than vaginal method when frozen-thawed semen was used, but its efficiency was not as high as laparoscopic method. Also, SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects of EY.

Effect of sperm concentration on characteristics and fertilization capacity of rooster sperm frozen in the presence of the antioxidants catalase and vitamin E.

The objective of this study conducted was to determine the influence of different levels of sperm concentration, including catalase (CAT) and vitamin E (VitE) in rooster semen extender on postthawed quality and fertility of rooster semen. Semen was collected twice a week from six roosters (Arian) and diluted according to experimental treatments consisting of sperm suspensions containing different sperm concentrations (200, 400, and 600 × 106 sperm/mL) without antioxidant supplementation as control (Con) groups (Con200, Con400, and Con600, respectively), sperm suspensions containing different sperm concentrations (200, 400, and 600 × 106 sperm/mL) supplemented with 5-µg/mL VitE (VitE200, VitE400, and VitE600, respectively) and different sperm concentrations (200, 400, and 600 × 106 sperm/mL) supplementation with 100 IU/mL CAT (CAT200, CAT400, and CAT600, respectively). After thawing; sperm motility, membrane integrity, and mitochondrial function were assessed. Fertility and hatchability rates were determined by using 100 artificially inseminated hens. The percentage of total motility (TM) and activity of mitochondria decreased (P < 0.05) as the sperm concentration increased in control groups. So, the lowest percentage of the TM and activity of mitochondria were observed in the Con600 as compared with other treatment groups. Extenders containing 100 IU/mL CAT and 5-µg/mL VitE resulted in higher (P < 0.05) TM, progressive motility, membrane integrity, and activity of mitochondria compared with control groups. Adding VitE and CAT in different sperm concentrations, the percentage of TM, membrane integrity, and activity of mitochondria decreased (P < 0.05) as the sperm concentration decreased. The highest (P < 0.05) membrane integrity, TM, and progressive motility were recorded at VitE400 and CAT400. Including VitE and CAT in rooster extender with different level sperm concentrations had no effect (P > 0.05) on fertility and hatchability rates. In conclusion, although adding VitE and CAT in extender with different levels of sperm concentration improved postthawed quality of rooster semen, but adding VitE and CAT in the extender have no effect on fertility rate.

Effect of various concentrations of butylated hydroxyanisole and butylated hydroxytoluene on freezing capacity of Turkman stallion sperm.

The present study aimed to determine the effect of different concentrations of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on post-thaw stallion sperm quality. The ejaculates collected from four healthy mature Turkmen stallions were pooled and divided into eight aliquots. The samples were diluted with extenders containing different concentrations (0.5, 1 or 2mM/mL) of BHA or BHT. The positive control (PC) samples were diluted with extender containing 0.5% ethanol (v/v) whereas; the negative control (NC) samples were diluted with basic extender only. Semen samples were frozen according to a standard protocol. After thawing of samples, sperm motility, viability, membrane integrity, total abnormality and lipid peroxidation were assessed. The greatest (P<0.05) values for total sperm motility, viability and plasma membrane functionality and least values for malonedialdehyde (MDA) concentration were observed in samples supplemented either with 1mM BHT or 2mM BHA. However, the progressive motility was greater (P<0.05) only in samples treated with 2mM BHA. In conclusion, the use of 1mM BHT or 2mM BHA in extender improves the freezing capacity of stallion sperm by reducing oxidative stress during freeze-thaw process.

Are the optimum levels of the catalase and vitamin E in rooster semen extender after freezing-thawing influenced by sperm concentration?

To date, there has no report to evaluate the interaction effects of antioxidant and sperm concentration in rooster semen cryopreservation. This study was aimed to investigate the effects of vitamin E (VitE) and catalase (CAT) at different sperm concentrations on the rooster post-thawed sperm quality. Semen samples were collected twice a week from ten roosters (ROS 308) and diluted according to experimental treatments. The treatments consist of different sperm concentrations (200, 400 and 600 × 106 sperm/mL) with supplementation VitE (5 µg/mL; VitE200, VitE400, and VitE600, respectively) or CAT (100 IU/mL; CAT200, CAT400, CAT600, respectively) and without antioxidants [Control (Con); Con200, Con400, Con600, respectively]. After thawing, motion characteristics were assessed using a CASA system. Plasma membrane integrity and malondialdehyde (MDA) level were evaluated with Hypoosmotic swelling test (HOST) and Thiobarbituric acid (TBA), respectively. The higher percentage of total motility, progressive motility, viability and membrane integrity were obtained in VitE400 (81.16 ± 1.21, 18.44 ± 1.19, 85.47 ± 1.07, 86.91 ± 1.16, respectively) and CAT400 (79.38 ± 1.21, 17.19 ± 1.19, 83.42 ± 1.07, 85.73 ± 1.16, respectively) compared to control groups. Moreover, the lowest percentage of MDA was measured in VitE400, VitE600 and CAT400 rather than other groups (1.489, 1.500, 1.510 ± 0.06, respectively). In conclusion, the results of the present study demonstrate that VitE (5 µg/mL) and CAT (100 IU/mL) independently at sperm concentration, 400 million sperm/mL could beneficial effect for preservation of rooster semen during cryopreservation.

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.

Effects of dietary supplementation of pioglitazone on metabolism, milk yield, and reproductive performance in transition dairy cows.

The objective of this study was to investigate the effect of dietary supplementation of pioglitazone (PGT), a specific ligand for PPARγ, on metabolic dynamics, milk production, and reproductive performance of transition dairy cows. Eighty multiparous Holstein cows in their second or more lactations were blocked by the calving date and parity and assigned randomly to four dietary groups (n = 20 cow/treatment) including control (no PGT-/-), supplemented with PGT (6-mg PGT/kg body weight) from Day -14 to +21 relative to parturition (PGT+/+) or only during prepartum (PGT+/-) or postpartum periods (PGT-/+). Postpartum body condition score and body weight loss decreased (P < 0.05) in all PGT-supplemented groups. Milk yield was not affected by PGT supplementation (P > 0.05). Percentage of milk fat decreased (P < 0.05) in all PGT-treated groups; however, milk fat yield was lower (P < 0.05) in PGT (+/+) and PGT (+/-) groups compared with PGT (-/-). Peripartum (Day -7 to +7) concentrations of plasma nonesterified fatty acids and ß-Hydroxybutyrate decreased in PGT (+/+) but not in the PGT (-/-) group (P < 0.05). During the postpartum period, PGT reduced (P > 0.05) plasma concentrations of nonesterified fatty acids in all PGT-treated groups but did not affect ß-Hydroxybutyrate level. Plasma concentrations of triglycerides decreased in all PGT-supplemented groups. Supplementation of PGT increased the peripartum concentrations of plasma glucose in PGT (+/+) and PGT (+/-) groups compared with control. Plasma concentrations of insulin-like growth factor 1 were higher in PGT (+/+) compared with the control group during both the peripartum and postpartum periods. Plasma concentrations of growth hormone and insulin were not affected by PGT treatment (P > 0.05). Mean days to ovulation were lower in PGT (+/+) and PGT (-/+), and the proportion of cows ovulating by Day 14 postpartum was higher in PGT (+/+) compared with control. Days open were shorter in PGT (+/+), PGT (+/-), and PGT (-/+) groups compared with control. However, the proportion of pregnant cows at 120 days in milk were higher in all PGT-supplemented groups. The results showed positive effects of dietary supplementation of PGT, especially supplementation during both the prepartum and postpartum periods, on metabolic dynamics, ovarian function, and reproductive performance in transition dairy cows.

Comparative evaluation of Nabi and Beltsville extenders for cryopreservation of rooster semen.

Two experiments were conducted to evaluate the new rooster semen freezing extender which is containing a low level of glycerol and soybean lecithin as an alternative protective agent in the extender. The aim of the first experiment was to evaluate a new extender for freeze-thawing rooster semen known as "Nabi" extender compared to Beltsville. Second experiment was also performed to determine whether the Nabi extender has negative reactions on fertilization after artificial insemination (AI) or no. In the first experiment, post-thaw motion parameters, mitochondrial function and sperm apoptosis were analyzed using Sperm Class Analyzer (SCA), rhodamine-123 and Annexin-V, respectively for frozen-thawed semen in Nabi and Beltsville extender. Results showed that total motility, progressive motility, velocity parameters (VCL, VSL, VAP, LIN and STR) and live spermatozoa with active mitochondria were significantly higher in Nabi compare to Beltsville extender (P < 0.01). Also, the percentages of post-thawed live and early apoptotic spermatozoa were significantly higher in Nabi compared to Beltsville extender (14.46 ± 0.95 vs. 19.27 ± 0.95 and 14.83 ± 4.51 vs. 39.27 ± 4.51, respectively). For apoptotic spermatozoa, the percentages of post-thawed late apoptotic spermatozoa were significantly lower in Nabi (29.66 ± 3.11) compared to Beltsville extender (69.07 ± 3.11), but the type of extender had no effect on the percentages of post-thawed necrotic spermatozoa. In the second experiment, 20 broiler breeder hens (Ross 308) were inseminated with thawed semen using the new freezing diluents or fresh semen for determination of fertility rate. Fertility rate with thawed semen (with Nabi extender) was lower compared to fresh semen (by approximately 8% points). It can be concluded that Nabi extender would improve post-thawed rooster sperm in vitro quality compared to Beltsville extender. The fertility rates of insemination in hens with freeze-thaw sperm were comparable with fresh sperm.