I. Caballero

PSP-I/PSP-II spermadhesin exert a decapacitation effect on highly extended boar spermatozoa.

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma that is able to preserve, in vitro, the viability, motility and mitochondrial activity of highly-extended boar spermatozoa. However, a relationship between the protective effects of the heterodimer and sperm capacitation is still unclear. The present study investigated the effect of the PSP-I/PSP-II (1.5 mg/mL) on membrane stability, intracellular calcium concentration ([Ca(2+)](I)) and plasma membrane and acrosome integrity of highly extended boar spermatozoa. Boar spermatozoa were diluted to 1 x 10(6) spermatozoa/mL and incubated at 38 degrees C in Phosphate-buffered saline (PBS) for 10, 30, 60, 120 and 300 min or in modified Tris-buffered medium (mTBM) for 10, 20, 30, 60 and 120 min. After each incubation time, the membrane stability (using Merocyanine-540/Yo-Pro-1), elevation of [Ca(2+)](I) (using Fluo-3-AM/PI) and the sperm plasma membrane and acrosome integrity (using SYBR-14/PI/PE-PNA) were evaluated by flow cytometry. As expected, exposure of the spermatozoa to the PSP-I/PSP-II preserved the plasma membrane and acrosome integrity compared to non-exposed spermatozoa in both media PBS and mTBM (p < .01). The evaluation of membrane stability showed no differences in the percentages of viable sperm with instable plasma membrane in the presence of the PSP-I/PSP-II compared to controls irrespective of the dilution media. The evaluation of the [Ca(2+)](I) levels showed that while spermatozoa incubated in mTBM and exposed to PSP-I/PSP-II had lower [Ca(2+)](I) than controls (39.08% vs. 47.97%, respectively; p < .05), no differences were observed in those samples incubated in PBS. However, a temporal evaluation of the samples showed that a similar proportion of live spermatozoa were able to achieve high levels of [Ca(2+)](I) and membrane instability independent of the presence of PSP-I/PSP-II. In conclusion, PSP-I/PSP-II exert a non-permanent decapacitation effect on highly extended boar spermatozoa that is related with a delay in the increase of [Ca(2+)](I) levels.

Major proteins of boar seminal plasma as a tool for biotechnological preservation of spermatozoa

Boar seminal plasma is a complex mixture of secretions from the testes, epididymides, and the male accessory reproductive organs which bathe the spermatozoa at ejaculation. The seminal plasma contains factors, mostly proteins, which influence the spermatozoa, the female genital tract, and the ovum. In boars, most of the proteins belong to the spermadhesin family and bind to the sperm surface. Spermadhesins are multifunctional proteins with a wide range of ligand-binding abilities to heparin, phospholipids, protease inhibitors and carbohydrates; the family can be roughly divided into heparin-binding (AQN-1, AQN-3, AWN) and non-heparin-binding spermadhesins (PSP-I/PSP-II heterodimer). These proteins have various effects promoting or inhibiting sperm functions including motility, oviduct binding, zona binding/penetration, and ultimately fertilization. The complexity of the environmental signals that influence these actions have implications for the uses of these proteins in vivo and in vitro, and may lead to uses in improving sperm storage.

Low‐Dose Insemination in Pigs: Problems and Possibilities

Low-dose AI procedures are required by the pig industry to efficiently utilize emerging sperm technologies, such as cryopreservation and sex-sorting. Currently, several different procedures for inseminating with a low or very low number of spermatozoa have been described. Deep intrauterine insemination allows the deposition of the spermatozoa in the depth of the uterine horn, allowing a significant reduction in the number of spermatozoa inseminated with maintenance of optimal reproductive performance. Intra-oviductal laparoscopic insemination has been recently applied in pigs. This technique has proved to be applicable with diluted and sex-sorted spermatozoa. This review discusses several problems encountered during the development of deep intrauterine insemination and intra-oviductal laparoscopic insemination of pigs and provides potential solutions for the practical application of both the technologies.

Influence of seminal plasma PSP-I/PSP-II spermadhesin on pig gamete interaction

The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm-ZP binding.

Improving the efficiency of insemination with sex-sorted spermatozoa.

The sorting of X- and Y-chromosome-bearing spermatozoa by flow cytometry is nowadays one of the most apt assisted-reproduction technologies in livestock production. Potential economic and biological benefits, as well as those related to easier management of herds, have been reported arising out of the application of this technique, especially in cattle. Yet, the sex-sorting procedure induces damage to spermatozoa, affecting their function and fertilizing ability. Different species present varying degrees of susceptibility to damage from the sorting process and each has its own requirements for sex-sorted insemination procedures. Thus, several new protocols and strategies have been designed for the handling of sorted spermatozoa, with the main objective of optimizing their fertilizing ability and the consequent application of flow-cytometric sex-sorting technology. This article reviews current advances in this technology, pointing out the components to be improved before this technology may be widely applied in different domestic species.

In vitro postwarming viability of vitrified porcine embryos: effect of cryostorage length.

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN(2)) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN(2) has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.

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.

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex-sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex-sorted sperm frozen at low sperm concentrations (20 × 10(6) sperm/ml; S20 group). Non-sorted spermatozoa frozen at 1000 × 10(6) (C1000 group) and 20 × 10(6) (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post-thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non-sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post-thaw motility of sex-sorted spermatozoa frozen at low concentrations.

Pre-pubertal Di(2-ethylhexyl) Phthalate (DEHP) Exposure of Young Boars Did Not Affect Sperm In vitro Penetration Capacity of Homologous Oocytes Post-puberty

Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinylchloride (PVC) products (e.g., plastic bags and medical equipment), has been reported to have toxic effects on animal reproduction and is considered an environmental hazard based, mostly, on rodent studies. However, the doses used in these studies are often considerably higher than that presumed in human exposure. In the present study we used young boars as model animals to assess the effects of pre-pubertal DEHP exposure on the ability of spermatozoa to penetrate homologous oocytes in vitro. Eight pairs of cross-bred male boar siblings were used. One brother in each pair became, at random, the test animal exposed to DEHP per os, three times a week, from 3 to 7 weeks of age while the other acted as the control, i.e., placebo-exposed. Semen was collected and frozen between 8 and 9 months of age and stored until spermatozoa were evaluated for their ability to in vitro penetrate in vitro-matured homologous oocytes post-thaw. Both the penetration rate and the number of spermatozoa per oocyte were considered within expected ranges for frozen boar semen of good quality. Penetration rate did not significantly differ (p > 0.05) between the groups with DEHP-exposed: 50%; control: 59%, which could be owing to a large variation between boars, and between replicates. The number of spermatozoa in the ooplasm was low and similar (p > 0.05) between the groups with DEHP-exposed: 1.5 and the control: 1.7. Under the conditions of the present experiment, pre-pubertal exposure to DEHP does not seem to cause a deleterious effect on the in vitro fertilizing ability of frozen spermatozoa post-puberty.

70 Effect of cryoprotectant concentration on the in vitro survival and cell proliferation of porcine blastocysts vitrified using the open pulled straw system

The objective of the present project was to study the effect of the concentrations of ethylene glycol (EG) and dimethyl sulfoxide (DMSO) during vitrification on the survival and hatching rates of porcine blastocysts. Embryos were collected by laparotomy from weaned crossbred sows (n = 18), vitrified, and warmed (one-step dilution) as described by Cuello et al. (2004 Theriogenology 62, 1144–1152). Vitrification was performed in different concentrations of EG and DMSO (15%, 16%, and 17% v/v for each cryoprotectant) or in an EG-based medium (40% v/v) using superfine open pulled straws. Fresh and vitrified blastocysts were cultured for 24 h in TCM199 and assessed by stereomicroscopy during the culture. Blastocysts that reformed their blastocoelic cavities after warming, displaying a normal zona pellucida and excellent appearance, were considered viable. The in vitro survival rate was defined as the ratio of viable embryos divided by the total number of embryos cultured. The hatching rate is determined as the ratio of the number of embryos hatched in vitro to the total number embryos cultured. Some vitrified and fresh embryos classified as viable were processed for Hoechst 33342 staining and proliferation cell nuclear antigen (PCNA) inmunolocalization. The proliferation index was defined as the number of PCNA-positive nuclei divided by the total number of nuclei stained with Hoechst 33342. Data were analyzed by ANOVA using the MIXED procedure (SPSS version 11.5; SPSS, Inc., Chicago, IL, USA). Data were expressed as mean values ± SEM. The survival rate was similar for fresh and vitrified blastocysts, except for blastocysts vitrified using 15% cryoprotectants, which displayed a lower (P < 0.05) survival rate (84.2 ± 4.8%) than fresh blastocysts (94.6 ± 5%) and blastocysts vitrified using 40% EG, 16%, or 17% EG-DMSO (88.8 ± 4.9, 96.8 ± 4.9, and 96.4 ± 5%, respectively). Fresh blastocysts showed a higher (P < 0.05) hatching rate (80.7 ± 4.5%) than their vitrified counterparts (range: 48.4 ± 7.7–55.3 ± 7.8%). Vitrified and fresh blastocysts showed similar cell proliferation indexes (range: 75.8 ± 3.2–83.7 ± 3). When only hatched blastocysts among groups were compared, the proliferation rate decreased (P < 0.05) after vitrification with 17% EG-DMSO. Among vitrification groups, there was no significant difference in the number of total cells. However, vitrified blastocysts had a lower (P < 0.05) total cell number (range: 116.6 ± 6.7–124.8 ± 6.6) than fresh blastocysts (195.5 ± 11.4). In conclusion, under our experimental conditions, the concentration of EG-DMSO can be decreased until 16% in the vitrification medium with no reduction of the in vitro developmental ability of the blastocysts. In addition, a 40% EG-based medium can be used for vitrification with results similar to those achieved using a medium containing 16% EG-DMSO.