Papain and its inhibitor E-64 reduce camelid semen viscosity without impairing sperm function and improve post-thaw motility rates.

In camelids, the development of assisted reproductive technologies is impaired by the viscous nature of the semen. The protease papain has shown promise in reducing viscosity, although its effect on sperm integrity is unknown. The present study determined the optimal papain concentration and exposure time to reduce seminal plasma viscosity and investigated the effect of papain and its inhibitor E-64 on sperm function and cryopreservation in alpacas. Papain (0.1mg mL-1, 20min, 37°C) eliminated alpaca semen viscosity while maintaining sperm motility, viability, acrosome integrity and DNA integrity. Furthermore E-64 (10 µM at 37°C for 5min after 20min papain) inhibited the papain without impairing sperm function. Cryopreserved, papain-treated alpaca spermatozoa exhibited higher total motility rates after chilling and 0 and 1h after thawing compared with control (untreated) samples. Papain treatment, followed by inhibition of papain with E-64, is effective in reducing alpaca seminal plasma viscosity without impairing sperm integrity and improves post-thaw motility rates of cryopreserved alpaca spermatozoa. The use of the combination of papain and E-64 to eliminate the viscous component of camelid semen may aid the development of assisted reproductive technologies in camelids.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Birth of kids after artificial insemination with sex-sorted, frozen-thawed goat spermatozoa.

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX(®) ) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids.

Glycosaminoglycans in the accessory sex glands, testes and seminal plasma of alpaca and ram.

The viscous nature of alpaca semen limits its use in cryopreservation and other assisted reproductive technologies. The cause and source of this viscosity is unknown although it has been postulated, but never proven, that glycosaminoglycans (GAGs) secreted by the bulbourethral gland are responsible. The present study investigated the concentration and composition of GAGs in alpaca seminal plasma, testes, bulbourethral gland and prostate gland and compared them to those in the ram to determine the relationship between seminal plasma GAGs and viscosity and to identify the source of seminal plasma GAGs. Alpaca seminal plasma contained more GAGs than ram (P<0.001) and the predominant GAG, keratan sulfate, was correlated with viscosity (P=0.05, R(2)=0.2635). The alpaca bulbourethral gland contained most GAGs compared with prostate or testis (P<0.001). In the ram, the prostate contained most GAGs. These findings suggest that GAGs, particularly keratan sulfate, may be the cause of seminal plasma viscosity in alpacas, and that the seminal plasma GAGs originate from the bulbourethral gland.

ß-Nerve growth factor is a major component of alpaca seminal plasma and induces ovulation in female alpacas.

Ovulation in camelids is induced by an unidentified protein in the seminal plasma of the male termed 'ovulation-inducing factor'. This protein has been reported to be a 14-kDa protein under reducing conditions, which, when purified from seminal plasma, induces ovulation in llamas. The identification of this protein and investigation of its potential to induce ovulation in camelids may aid the development of protocols for the induction of ovulation. In the present study, alpaca seminal plasma proteins were separated using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the most abundant protein of 14 kDa was identified as ß-nerve growth factor (ß-NGF) by liquid chromatography mass spectrometry. Female alpacas (n = 5 per group) were given intramuscular injections of: (1) 1 mL of 0.9% saline; (2) 4 µg buserelin, a gonadotrophin-releasing hormone agonist; (3) 2 mL alpaca seminal plasma; or (4) 1mg human ß-NGF. Ovulation was detected by transrectal ultrasonography 8 days after treatment and confirmed by plasma progesterone concentrations. Ovulation occurred in 0%, 80%, 80% and 80% of animals treated with saline, buserelin, seminal plasma and ß-NGF, respectively. Treatment type did not affect the diameter of the corpus luteum, but plasma progesterone concentrations were lower in saline-treated animals than in the other treatment groups owing to the lack of a corpus luteum. The present study is the first to identify the ovulation-inducing factor protein in alpacas. ß-NGF successfully induces ovulation in alpacas and this finding may lead to new methods for the induction of ovulation in camelids.

Seminal plasma components in camelids and comparisons with other species.

Camelid semen is characterized by a highly viscous, low-volume ejaculate with a low concentration of spermatozoa that exhibit low progressive motility. The viscous seminal plasma is currently the major impediment to the development of assisted reproductive technologies (ARTs) in camelids. To advance ARTs such as sperm cryopreservation and artificial insemination in camelids, it is necessary to identify the cause of the viscosity and gain an understanding of the role of seminal plasma components on sperm function and fertility. Numerous compounds and proteins have been identified as mediators of sperm function and predictors of fertility in other livestock species, and understanding the importance of specific proteins has progressed the success of ARTs in these species. Current knowledge on the components of camelid seminal plasma is outlined, together with the implications of these components for the development of ARTs in camelids. The cause of semen viscosity, as well as proteins that are present in camelid seminal plasma, is described for the first time. Seminal plasma components are compared with those of other species to hypothesize their role in sperm function and fertility.

Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation.

Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82(®) and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freezing media could be identified, suggesting that both cryodiluents would be suitable for incorporation into a sex-preselection protocol for stallion spermatozoa. Experiment 2 compared Kenney's modified Tyrode's (KMT) and Sperm TALP (Sp-TALP) as the staining and incubation medium for stallion spermatozoa prior to sex-sorting. A significant increase in the percentage of acrosome-reacted spermatozoa occurred after staining and incubation in the clarified Sp-TALP compared with KMT. As no improvements in sorting rates were achieved using Sp-TALP, it was concluded that stallion sorting protocols could include KMT as the staining and incubation medium while either INRA 82(®) or lactose-EDTA could be employed as a cryodiluents.

Seminal plasma proteins do not consistently improve fertility after cervical insemination of ewes with non-sorted or sex-sorted frozen-thawed ram spermatozoa.

The effect of supplementation of sex-sorted and non-sorted spermatozoa with seminal plasma protein (SPP) on fertility after cervical insemination was examined in the present study. Spermatozoa were sorted into high purity X and Y chromosome-bearing spermatozoa or not sorted and then either supplemented with SPP (>10 kDa) before freezing and/or after thawing (non-sorted only) or processed without supplementation. Inseminations were performed over 2 days with ewes receiving 100 or 25 million motile non-sorted spermatozoa in the cervix or uterus, respectively, or two cervical inseminations of 3.5 million motile sorted spermatozoa. Pregnancy rates in cervically inseminated ewes were unaffected by supplementation of sorted or non-sorted spermatozoa with SPP before freezing compared with no supplementation. The effect of post-thaw supplementation of non-sorted spermatozoa with SPP on pregnancy rates after cervical insemination varied with the day of insemination (P < 0.05); fertility was similar to laparoscopic insemination on Day 1 (56.0 +/- 10.2% v. 58.6 +/- 10.1%), but not on Day 2 (23.1 +/- 7.4% v. 66.7 +/- 9.2%). In conclusion, under the conditions of the present study, SPP did not consistently improve pregnancy rates after cervical insemination with frozen-thawed ram spermatozoa. This is the first report of pregnancies (5/56 ewes inseminated) after cervical insemination with frozen-thawed sex-sorted ram spermatozoa. Although the success rate is low, the findings are encouraging because ewes inseminated with the sex-sorted spermatozoa received only 7% of the recommended dose (100 million motile) for cervical insemination of frozen-thawed spermatozoa.

Evaluation of the function of fresh and frozen-thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay.

The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen-thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen-thawed non-sorted spermatozoa; and (4) frozen-thawed sex-sorted spermatozoa. Spermatozoa found in association with the zona pellucida of the bovine oocytes were deemed 'attached' or 'bound' depending on their characterisation as either acrosome intact or acrosome reacted, respectively. Significantly less frozen-thawed spermatozoa were found attached to the oocytes compared with fresh spermatozoa. No significant differences were identified between the number of attached sex-sorted and non-sorted frozen-thawed spermatozoa. However, significantly more sex-sorted than non-sorted fresh spermatozoa were found attached to the oocytes after 1 h coincubation, although after 3 h coincubation this difference was no longer apparent. In conclusion, sex-sorted fresh and frozen-thawed stallion spermatozoa are functionally capable of attaching and binding to bovine oocytes in vitro. Furthermore, fresh sex-sorted spermatozoa attach better than non-sorted spermatozoa, suggesting that they have a more advanced capacitation-like status.

Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase.

To determine whether flow sorting increased the susceptibility of spermatozoa to reactive oxygen species (ROS), ram semen was either diluted with Tris medium (100 x 10(6) spermatozoa mL(-1); D) or highly diluted (10(6) spermatozoa mL(-1)) before being centrifuged (DC) at 750g for 7.5 min at 21 degrees C or flow-sorted (S) before cryopreservation. Thawed spermatozoa were resuspended in graded concentrations of hydrogen peroxide to induce oxidative stress. In Experiment 1, following exposure to 30 or 45 muM hydrogen peroxide (H(2)O(2)), the total motility (%) of DC (41.0 +/- 7.3 or 25.7 +/- 6.7, respectively) and S spermatozoa (33.8 +/- 6.3 or 20.1 +/- 6.3, respectively) was lower (P < 0.001) than that of D spermatozoa (58.7 +/- 5.6 or 44.5 +/- 6.7, respectively). In Experiment 2, supplementation of samples containing H(2)O(2) with catalase (150 IU mL(-1)) or seminal plasma proteins (4 mg protein per 10(8) spermatozoa) negated oxidative stress, resulting in comparable values to samples receiving no H(2)O(2)in terms of the proportion of spermatozoa with stable plasmalemma (as determined using merocyanine-540 and Yo-Pro-1) in the D and S groups, the proportion of viable, acrosome-intact spermatozoa (as determined by fluorescein isothiocyanate and propidium iodide staining) in the D group and the motility of control (undiluted) and S spermatozoa. Neither H(2)O(2) nor sperm type (i.e. D, DC or S) had any effect on intracellular concentrations of ROS. These results show that flow sorting increases the susceptibility of spermatozoa to ROS, but the inclusion of anti-oxidants or seminal plasma as part of the sorting protocol improves resistance to oxidative stress.

Quality parameters for alpaca (Vicugna pacos) semen are affected by semen collection procedure.

Artificial insemination (AI) is poorly developed in camelids owing to the difficulty in collecting high quality semen and the highly viscous nature of the semen. Semen collected by artificial vagina (AV) is often of low quality and must be improved before any further development of AI technology can occur. The present study investigated the effects of adding a cervix-like stricture to the AV, presence of females, collecting semen into Androhep, skim-milk or Tris diluents, and catalase supplementation (0, 100, 200 or 600 units/ml) of Tris diluent on alpaca semen quality parameters. The addition of a cervix-like stricture increased mating length (p < 0.05), whilst the presence of females during semen collection did not improve semen quality parameters (p > 0.05). Collection of semen into Tris diluent improved sperm motility (58.0 +/- 11.9%) compared with the control (34.0 +/- 10.8%; p < 0.05), Androhep (33.5 +/- 10.7%) and skim-milk diluents (28.2 +/- 10.4%). Semen viscosity was reduced by collection into Androhep (4.6 +/- 1.7 mm) and skim-milk diluents (3.6 +/- 1.3 mm) compared with Tris diluent (5.7 +/- 2.1 mm) and no collection medium (9.3 +/- 3.5 mm; p < 0.05). Tris diluent supplemented with 100, 200 or 600 units/ml catalase increased semen viscosity (5.0 +/- 3.2 and 4.9 +/- 3.2 mm). Collection of alpaca semen by AV into Tris diluent increased semen quality facilitating further development of AI technology in alpacas.

Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze-thaw damage.

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n=four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37 degrees C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze-thawing.

Sperm sexing in sheep and cattle: the exception and the rule.

Flow cytometric sorting for the preselection of sex has progressed considerably in the 20 years since its inception. This technique has allowed the production of pre-sexed offspring in a multitude of species and become a commercial success in cattle around the world. However, due to the stress inherent to the sex-sorting process, sex-sorted spermatozoa are widely recognized as functionally compromised in terms of their fertilizing lifespan within the female reproductive tract as a result of reduced motility and viability and changed functional state. These characteristics, when compared to non-sorted controls, are manifest in vivo as lower fertility. However, improvements to the technology and a greater understanding of its biological impact have facilitated recent developments in sheep, showing sex-sorting is capable of selecting a functionally superior population in terms of both in vitro and in vivo function. These results are reviewed in the context of recent developments in other species and the reasons for success after artificial insemination with sex-sorted ram spermatozoa are discussed.

The effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram sperm.

The objective was to determine the optimum timing of insemination and minimum effective dose rate of sex-sorted ram sperm. Semen from three Merino rams was sorted into high purity X- and Y-chromosome bearing sperm populations. Ovulation was controlled in 732 Merino ewes using PMSG at progestagen pessary removal and GnRH 36h later. Sorted (S) and non-sorted (NS) doses of 1 or 15x10(6) motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66h after progestagen withdrawal. An additional treatment dose of 0.5x10(6) S or NS sperm was inseminated at the 58h time point (n=60). Pregnancy was diagnosed by ultrasound at 60-62 d gestation. Both 1x10(6) and 15x10(6) sperm achieved similar pregnancy rates, regardless of sperm type, at 58h (S1: 46+/-9.4%; S15: 43+/-9.3%; NS1: 41+/-9.2%; NS15: 49+/-9.4%). However, pregnancy rates were lower (P<0.05) for doses of 1 than 15x10(6) sperm inseminated at 50 (15+/-6.3% vs. 36+/-9.1%), 54 (14+/-4.4% vs. 55+/-7.3%), 62 (33+/-6.9% vs. 54+/-7.3%), and 66h (29+/-8.6% vs. 56+/-9.5%). There was no difference between S and NS sperm for inseminations with 0.5x10(6) motile sperm at 58h after PR (15+/-3.6% vs. 14+/-3.3%), nor with 15x10(6) motile sperm at all insemination times (49+/-6.3% vs. 49+/-6.3%). However, fertility was higher for S than NS sperm at the 1x10(6) dose level (37+/-6.1% and 16+/-4.0%). More than 90% of lambs born were of the predicted sex. We hypothesise that the sorting process selects a homogeneous, fertile sub-population of sperm, removing those that are dead, damaged and morphologically abnormal.

Development of procedures for sex-sorting frozen-thawed bovine spermatozoa.

Dairy bull sperm may be sex-sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well-managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex-sorting from frozen-thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex-sort and re-freeze frozen-thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm or BoviPure, followed by staining in one of three diluents (Androhep, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen-thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 +/- 0.6% vs 19.5 +/- 2.4%; p = 0.003) after centrifugation in PureSperm rather than BoviPure gradients. Sperm orientation (p < 0.05) and motility (69.9 +/- 3.0 vs 55.6 +/- 4.0; p < 0.001) were highest after staining in Androhep rather than in TALP buffer. Sperm were more motile (58.2 +/- 4.7 vs 38.7 +/- 3.5; p < 0.001) and had better acrosome integrity (74.3 +/- 2.9 vs 66.8 +/- 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen-thawed bull sperm to be sex-sorted with high resolution between the sexes, then re-frozen and thawed with retention of motility and acrosome integrity.

Germany/Australia index of sperm sex sortability in elephants and rhinoceros.

Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.

Early pregnancy loss in sows after low dose, deep uterine artificial insemination with sex-sorted, frozen-thawed sperm.

Recent developments in reproductive technologies have enabled the production of piglets of a predetermined sex via non-surgical, low dose artificial insemination. The practical application of sex-sorting technology to the pig is made challenging by the large numbers of sperm required for successful insemination of sows. One way of overcoming the time required for sex-sorting may be to create a bank of cryopreserved, sex-sorted sperm, thus making available appropriate doses as sows require insemination. To date, little success has been achieved with non-surgical inseminations of sex-sorted boar sperm. This study attempted to achieve litters of a predetermined sex after a double insemination of sows with 160x10(6) sex-sorted, frozen-thawed sperm. Sows were synchronised and sperm were non-surgically inseminated into the proximal third of the uterine horn at 36 and 42 h after hCG administration. Sows inseminated with sex-sorted sperm achieved similar pregnancy rates to those receiving an equal dose of unsorted, frozen-thawed sperm. However, all sows conceiving after insemination with sex-sorted sperm returned to oestrus within 57 days of insemination. This was a higher rate of pregnancy loss than observed for sows inseminated with unsorted sperm (37.5%; P=0.031). A combination of low sperm numbers and potentially compromised developmental capability of embryos derived from sex-sorted sperm may have resulted in this early stage loss of pregnancy.

Field fertility of frozen-thawed boar sperm at low doses using non-surgical, deep uterine insemination.

The lowest dose of frozen-thawed boar sperm used for deep uterine artificial insemination (DUI) of sows has been 100x10(6). A three stage field study was performed to establish to what level the dose of frozen-thawed sperm used for DUI could be reduced without adversely affecting the fertility of the sow. In stage 1, 15 sows were inseminated twice with 1000x10(6) fresh or frozen-thawed sperm at 24 and 36 h post-detection of oestrus. In stage 2, 262 sows were inseminated with 62.5, 250 or 1000x10(6) fresh or frozen-thawed sperm at 24, 36, or 24 and 36 h after detection of oestrus. Stage 3 involved post mortem investigation of the uterine lining to assess damage caused by insertion of the insemination catheter. All sows inseminated in stage 1 of the study farrowed. In stage 2, the non-return (NRR) and farrowing rates of each group were compared to a control double cervical insemination of 3250x10(6) fresh sperm. As few as 62.5x10(6) fresh sperm could be deposited at a single insemination without reduction in NRR or farrowing rates compared with the control group. A double DUI with 250x10(6) frozen-thawed sperm was required before fertility was equivalent to the controls. Investigation of the uterine lining after insertion of the DUI catheter revealed evidence of bleeding, warranting further investigation of the viability of widespread use of the Firflex catheter, despite the promising fertility achieved here with low doses of spermatozoa.

New aspects of boar semen freezing strategies.

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.

Application of seminal plasma in sex-sorting and sperm cryopreservation.

Substantial dilution of boar semen during processing decreased the concentration of seminal plasma, perhaps contributing to the decline in sperm quality after cryopreservation and sex-sorting. Results of replacing seminal plasma in investigations from many laboratories have been contradictory. Results and discussion here suggest that whereas membrane status can be influenced by seminal plasma, the action of its various components, both positive and negative, is determined in part by the membrane status of the spermatozoa to which it is being exposed. Although progress has been made in identifying components of seminal plasma responsible for its protective effect (notably PSP-I/II spermadhesin for sex-sorted boar spermatozoa), little is known (in any species) regarding how external factors may influence their levels, and their functionality, in seminal plasma. It is noteworthy that seminal plasma is beneficial to post-thaw quality of sex-sorted ram spermatozoa only when added before freezing, not after thawing. Therefore, the action of seminal plasma and its components is dependent on sperm-related factors, in particular the type of processing to which they have been previously exposed. Further research is needed to unravel these biological complexities, and then characterise and synthesise useful proteins within seminal plasma.