Exogenous ascorbic acid enhances vitrification survival of porcine in vitro-developed blastocysts but fails to improve the in vitro embryo production outcomes.

In this study, the effects of addition of the antioxidant ascorbic acid (AsA) were evaluated during porcine in vitro embryo production (IVP) and vitrification. In experiment 1, the effects of AsA supplementation during IVM, IVF and IVC were evaluated, using a total of 2744 oocytes in six replicates. The IVM, IVF and embryo IVC media were supplemented or not (control) with 50 µg/mL AsA in all possible combinations. No significant effects of AsA were detected in any of the maturation, fertilization or embryo development parameters assessed. In experiment 2, we evaluated the effects of adding AsA to vitrification-warming media on the post-warming survival and quality of blastocysts. Day-6 in vitro-produced blastocysts (N = 588) from six replicates were randomly divided in two groups, with vitrification and warming media either supplemented with 50 µg/mL AsA (VW + group) or un-supplemented (VW- control). Addition of AsA increased (P < 0.05) blastocyst survival rate after vitrification compared with that of VW- control embryos. Vitrification and warming increased (P < 0.05) the production of oxygen species (ROS) and reduced (P < 0.05) the glutathione levels in blastocysts. Although VW + blastocysts displayed higher (P < 0.05) ROS levels than those of fresh control blastocysts, the levels were lower (P < 0.05) than those found in VW- control blastocysts. In conclusion, under the experimental conditions, supplementation of IVM/IVF/IVC media with AsA did not improve the embryo production in vitro. By contrast, the addition of AsA to chemically defined vitrification and warming media increased the survival of in vitro-produced porcine blastocysts by decreasing ROS production.

Feed intake and sperm morphology in Ogaden bucks supplemented with either agro-industrial by-products or khat (Catha edulis) leftover.

In this study, feed intake and sperm morphology were evaluated in Ogaden bucks supplemented with either agro-industrial by-products or khat leftover. Thirty-five bucks at about 1 year of age, and 15 +/- 1.5 kg initial body weight, were involved in a 12-week feeding programme that had four diet groups. The control (C) diet comprised grass hay alone, fed ad libitum; treatment 1 (T1) comprised grass hay ad libitum supplemented with a mix of agro-industrial by-products at 1% of body weight (BW); treatment 2 (T2) comprised grass hay ad libitum supplemented with khat leftovers at 1% of BW; and treatment 3 (T3) comprised khat leftover alone, fed ad libitum. Bucks in T1-T3 consumed the highest (p < 0.001) dry matter (DM), crude protein (CP), organic matter (OM) and gross energy (GE) compared with control. Among the treatment groups, DM, OM and GE intakes were higher (p < 0.05) in T3 and T2, while CP intake was highest (p < 0.05) in T1. T1 and T3 improved (p < 0.001) the percentage of morphologically-normal spermatozoa in comparison with C, with bucks in T3 being best. The proportion of total abnormal sperm head shapes and proximal cytoplasmic droplets was lowest (p < 0.01) in T1 and T3 and highest in C. Although feeding with T1 improved feed intake and sperm morphology, feeding with T3 showed the highest response. Thus, we conclude that T3 and T1 could be utilized as feedstuff to improve sperm morphology in goats under the smallholder farming system in Ethiopia.

Effect of Supplementation with Agro-industrial By-products and Khat (Catha edulis) leftovers on testicular growth and sperm production in Ogaden bucks.

The aim of this study was to characterize growth and sperm production parameters in Ogaden bucks fed a basal diet of hay and supplemented with agro-industrial by-products and Khat leftovers in Ethiopia. Thirty-five bucks with a mean (+/-SD) initial live body weight (BW) of 15.5 +/- 1.5 kg were randomly assigned to one of four dietary treatments for a period of 13 weeks. Treatments consisted of native hay fed ad libitum (control; C), native hay supplemented with a 1% of BW agro-industrial by-products (treatment 1; T1), native hay supplemented with Khat (Catha edulis) leftovers at a rate of 1% of BW (treatment 2; T2) and Khat leftovers fed ad libitum (treatment 3; T3). Bucks fed on T1-T3 had higher BW, body condition score, scrotal circumference (SC), testicular width and testicular length, compared to controls (P < 0.05). Also, bucks in T1-T3 had higher sperm progressive motility, sperm concentration per ml and total number of spermatozoa per ejaculate compared to controls (P < 0.05). Between treatments, bucks in T3 recorded the highest BW (17.2 +/- 0.16) and testicular size (21.1 +/- 0.17 cm). Both testicular and epididymal weight and dimensions were significantly affected (P < 0.05) by supplementation compared to controls. Testicular size was positively correlated to live BW (r = 0.53, P < 0.001). SC was positively correlated with ejaculate volume (r = 0.37, P < 0.001), sperm mass activity (r = 0.65, P < 0.001) and individual sperm progressive motility (r = 0.40; P < 0.001). Supplementation with Khat leftovers induced the highest improvement in live BW, testicular size, semen production and sperm motility in Ogaden bucks and can possibly be considered as a feed supplement to enhance goat production under smallholder livestock farming system in Ethiopia.

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture.

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate.

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.

Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan.

In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.

Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen.

Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800 x 10(6) cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800 x 10(6) cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310 g/20 min). Extender C was based on TRIS-citrate-fructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/P1), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (x 10(6)/straw) differed (P<0.001) between P1 (mean+/-SD, 138.1+/-14.8) and P2 (216.5+/-13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers x 10(6) in the Aldose of subjective motility (87.2+/-5.1 vs 125.3+/-5.1; P<0.05), linearity (70.6+/-4.3 vs 79.8+/-4.3; NS), intact-membrane (77.4+/-5 vs 108.5+/-5.1; P<0.001), and uncapacitated (36.5+/-2.5 vs 46.5+/-2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (x 10(6)/straw) were lower in P1 than in P2 for subjective motility (80.8+/-5.4 vs 92.0+/-5.4; NS), linearity (63.3+/-5.6 vs 73.1+/-5.6; NS), membrane integrity (77.7+/-3.6 vs 101.0+/-3.6; P<0.001), and uncapacitated spermatozoa (28.3+/-3.24 vs. 4.1+/-3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38 degrees C (44.0+/-2.4 vs 36.2+/-2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7+/-2.2 vs 34.4+/-2.3; P<0.001), subjective motile (57.5+/-2.7 vs 43.8+/-2.7; P<0.01), and linear motile (46.5+/-2.8 vs 33.7+/-2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6+/-1.5 vs 51.7+/-1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).

Fine structure of bovine blastocysts developed either in serum-free medium or in conventional co-culture with oviduct epithelial cells.

The ultrastructure of day 7 bovine blastocysts developed in vitro in either of two culture systems was compared with that of morphologically normal blastocytes collected non-surgically from superovulated cows on day 7 (Day 0 = day of insemination). The in vitro-embryos were obtained after culture of in vitro-matured and -fertilized oocytes either in a serum-free, cell-free medium (SFM, i.e. TCM 199 supplemented with BSA (10 mg/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml) and selenium (5 ng/ml) or in a serum-supplemented medium (TCM 199 and 10% (v/v) oestrous cow serum) together with bovine oviduct epithelial cells (BOEC). Five of the 8 blastocysts developed in SFM fulfilled the criteria set for normal morphology of the in vivo-developed blastocytes. In contrast, 6 out of 8 blastocysts developed in co-culture with BOEC were classified as morphologically deviated, and only 2 reached the criteria for morphological normality. In vitro-developed blastocysts with deviated morphology showed a higher degree of cytoplasmic vacuolation, short, less developed cell-to-cell contacts between trophoblast as well as between inner cell mass (icm)-cells, less developed apical microvilli on the trophoblast and wide inter-cellular spaces. Additionally, numerous cytoplasmic vesicles, phagosomes, lipid droplets and hooded mitochondria were commonly present, both in trophoblast and in icm-cells. The results indicate that a high proportion of blastocysts developed in co-culture with BOEC were morphologically deviated compared to those cultured in medium where serum and somatic cells were replaced by BSA, insulin, transferrin and selenium.

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability.

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.

A simple, rapid method for differential cell counts in porcine mammary secretions.

The use of the fluorescent dye acridine orange for making differential cell counts in mammary secretions from sows was investigated, and the variations in cell type during the lactation period were also studied. In untreated samples of mammary secretions polymorphonuclear leucocytes, mononuclear phagocytes, lymphocytes and epithelial cells could be identified with certainty, and the methodological error was small (1.80 to 2.22 per cent). The mammary secretions could be stored at 4 degrees C for six hours without any effect on the differential count. Washing the secretions decreased the percentage of polymorphonuclear leucocytes and increased the percentage of epithelial cells. The polymorphonuclear leucocytes predominated in colostrum (58.0 to 65.5 per cent) and epithelial cells predominated in milk (60 to 89 per cent). Polymorphonuclear leucocytes were the predominant phagocytes in all mammary secretions (7.6 to 65.5 per cent).