Pharmacological approaches to induce follicular growth and ovulation for fixed-time artificial insemination treatment regimens in ewes.

In Experiment I, during the non-breeding season, after intravaginal devices containing progesterone (P4) were withdrawn (n = 28), estrous rates were greater with treatment with 400 IU eCG (P < 0.05) than with FSH (10 and 15 mg) and no treatment. During the breeding season (n = 147), estrous and pregnancy rates after fixed-time artificial inseminations (FTAI) were similar among groups: 300 IU eCG; 10 mg FSH; and control (P > 0.05). In Experiment II (non-breeding season), ewes of one group were treated with 300 IU eCG (n = 8) and of two groups were treated with 10 mg FSH. In one FSH group, 250 µg estradiol benzoate (EB) were administered after 24 h (n = 9); in the other, 4 µg GnRH were administered after 36 h (n = 10). Serum P4 concentrations were greater in eCG-treated ewes (P < 0.05). Estrous rates were similar for the eCG- and FSH plus EB-treated ewes (P > 0.05). In Experiment III (breeding season), the treatments were: 300 IU eCG; 250 µg estradiol cypionate; 250 µg EB; or control (n = 22). Follicular growth was greater for eCG-treated ewes within 0-24 h and for control ewes within 48-72 h (P = 0.001). Although estrous and ovulation rates did not differ (P > 0.05), all eCG-treated ewes had ovulations. During the non-breeding season, FSH treatment promoted follicular growth but did not induce ovulations. For FTAI regimens, eCG was more effective than FSH plus GnRH and estradiol esters in inducing estrus and ovulation.

New insights and current tools for genetically engineered (GE) sheep and goats.

Genetically engineered sheep and goats represent useful models applied to proof of concepts, large-scale production of novel products or processes, and improvement of animal traits, which is of interest in biomedicine, biopharma, and livestock. This disruptive biotechnology arose in the 80s by injecting DNA fragments into the pronucleus of zygote-staged embryos. Pronuclear microinjection set the transgenic concept into people's mind but was characterized by inefficient and often frustrating results mostly because of uncontrolled and/or random integration and unpredictable transgene expression. Somatic cell nuclear transfer launched the second wave in the late 90s, solving several weaknesses of the previous technique by making feasible the transfer of a genetically modified and fully characterized cell into an enucleated oocyte, capable of cell reprogramming to generate genetically engineered animals. Important advances were also achieved during the 2000s with the arrival of new techniques like the lentivirus system, transposons, RNA interference, site-specific recombinases, and sperm-mediated transgenesis. We are now living the irruption of the third technological wave in which genome edition is possible by using endonucleases, particularly the CRISPR/Cas system. Sheep and goats were recently produced by CRISPR/Cas9, and for sure, cattle will be reported soon. We will see new genetically engineered farm animals produced by homologous recombination, multiple gene editing in one-step generation and conditional modifications, among other advancements. In the following decade, genome edition will continue expanding our technical possibilities, which will contribute to the advancement of science, the development of clinical or commercial applications, and the improvement of people's life quality around the world.

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.

Detection of endoplasmic reticulum stress markers and production enhancement treatments in transgenic goats expressing recombinant human butyrylcholinesterase.

Compromised lactation physiology has been observed in transgenic animals, possibly due to the excessive demand placed by the expression of complex recombinant glycoproteins in the mammary gland. In previous studies we described lactation parameters and milk composition characteristics of transgenic goats expressing recombinant human butyrylcholinesterase in milk, and we showed evidence suggesting that lactation cessation could be associated with endoplasmic reticulum stress. We now report data from immunohistochemistry studies targeting activation transcription factor 6 and caspase 12, two signal transducers associated with endoplasmic reticulum stress, designed to further elucidate potential mechanisms responsible for the disruption in mammary epithelium function previously described. We found strong evidence of endoplasmic reticulum stress associated with the premature cessation of lactation. In addition, we utilized previously generated knowledge to design and test two treatments for enhanced productivity in transgenic goats. Pre-partum treatment with reserpine and dexamethasone to stimulate mammary priming for lactation resulted in a significant increase in milk production on day 1 (573 ± 350 vs. 93 ± 92 mL; P < 0.01), first week (8,832 ± 2,286 vs. 5,946 ± 2,039; P < 0.01) and the first month of lactation (42.5 ± 10 vs. 34.9 ± 6 kg; P < 0.05) compared to untreated controls. Mammary infusions with inosine during the early stages of lactation to promote mammary stem-cell proliferation also resulted in significantly increased milk production volumes, ranging from 26 to 200% more milk, in the treated glands compared to placebo.

Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase.

The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, beta-lactoglobulin and alpha-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of alpha-lactalbumin was determined using the IDRing assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 +/- 0.97 vs. 2.24 +/- 0.25 mg/ml, P < 0.01), but was not different from non-transgenic controls by day 30 (0.99 +/- 0.46 vs. 0.90 +/- 0.11 mg/ml, P > 0.05). We concluded that a decreased/delayed expression of the alpha-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.

Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value.

The efficiency of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) in the propagation of aged goats with poor reproductive performance was evaluated in the present study. Follicular development was stimulated in donor goats with 80 mg follicle-stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. In addition, goats were heat synchronised with intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days and a luteolytic injection of 125 microg cloprostenol 36 h before sponge removal and LOPU. Following in vitro maturation (IVM), oocytes were fertilised in vitro with frozen-thawed semen produced using the egg yolk-free Bioxcell extender (IVM, L'Aigle, France). The average number of follicles aspirated (17.9 +/- 8.0 per goat), oocytes recovered (15.7 +/- 8.4 per goat) and cleavage after IVM/in vitro fertilisation followed by a short 24-h in vitro culture in modified synthetic oviduct fluid medium (72 +/- 7%) were similar to results reported previously by our group and others in younger goats. A total of 296 embryos was transferred into 50 heat-synchronised recipients, of which 40 became pregnant (80%) and 38 progressed all the way to term, delivering 86 live kids. The present study indicates that LOPU-IVEP can be used successfully to extend the reproductive life of valuable goats that have acquired difficulties becoming pregnant by artificial insemination after multiple kiddings.

State of the art in the production of transgenic goats.

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

Advanced assisted reproduction technologies (ART) in goats.

Assisted reproduction technologies (ART) are reviewed with special emphasis on goat genetic improvement programs. Estrous synchronization and artificial insemination are the most commonly used ART worldwide because of their simplicity and excellent cost/benefit, especially when proven sires are used. Multiple ovulation and embryo transfer (MOET) has not become widely used due to its unpredictability. In vitro embryo production using oocytes collected by laparoscopy from valuable donors has the potential to improve the results obtained from MOET and expand its applications (for example, using prepubertal donors). However, the costs and inefficiencies of the system might restrict its use to special situations. Finally, transgenesis and cloning are expected to have a significant impact on the future genetic improvement of livestock. However, because of low efficiencies and high costs, their present use is restricted to applications with high returns such as the production of recombinant proteins of pharmaceutical and biomedical interest.

Prepubertal propagation of transgenic cloned goats by laparoscopic ovum pick-up and in vitro embryo production.

The use of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production was evaluated in the early propagation of cloned goats. Ten kinder goats produced by somatic cell nuclear transfer technology were used as oocyte donors. Half of the donor animals were subjected to LOPU at 2-3 months of age (prior to induction of lactation), whereas the other five goats were subjected to LOPU at 6-7 months of age (following induction to lactation). They were stimulated with 80 mg NIH-FSH-P1 (Folltropin, Vetrepharm, Canada) together with 300 IU eCG (Novormon, Vetrepharm, Canada) administered intramuscularly 36 h prior to LOPU. The number of follicles aspirated and oocytes recovered was higher in the younger group of donors (57 +/- 7 and 41 +/- 4 vs. 28 +/- 2 and 25.8 +/- 2, p < 0.05), however, oocytes from animals in the late prepubertal age showed higher developmental capacity resulting in higher transferable embryo yield (81.4% vs. 67.8%, p < 0.01), pregnancy rate (80% vs. 40%, p < 0.05) and total kids born (27 vs. 15, p < 0.01). In conclusion, LOPU in combination with in vitro embryo production techniques is an efficient method for the early propagation of valuable goats produced by somatic cell nuclear transfer.

Effects of repetition, interval between treatments and season on the results from laparoscopic ovum pick-up in goats.

The present study was conducted to evaluate the follicular response and oocyte yield following repeated gonadotrophin stimulation and laparoscopic aspiration in goats and to assess the effects of the time interval between procedures and season. A total of 98 adult goats were subjected to laparoscopic ovum pick-up (LOPU) five consecutive times in a transgenic production programme. Oestrus was synchronised by means of intravaginal sponges inserted for 10 days coupled with 125 microg cloprostenol 36 h before sponge removal and LOPU, and follicular development was stimulated with 80 mg follicle stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. No difference was detected in the response for LOPUs 1, 2, 3 and 4. Although a small decrease in response was detected at LOPU 5 (P < 0.05), the numbers of follicles aspirated and oocytes recovered were not different from those at LOPU 1 and LOPUs 1 and 4, respectively. With respect to time interval between LOPU and season, all intervals and seasons produced acceptable responses, with no difference in follicles aspirated and oocytes recovered between intervals and seasons. These results indicate that LOPU may be repeated up to five times in goats at different intervals and in different seasons with little or no important change in overall response.

The in vitro and in vivo development of goat embryos produced by intracytoplasmic sperm injection using tail-cut spermatozoa.

The objective of this study was to assess the efficacy of a novel intracytoplasmic sperm injection (ICSI) procedure, as well as the in vitro and in vivo developmental competence of goat embryos produced by ICSI. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh goat semen was used for ICSI following Percoll gradient washing. Tail-cut spermatozoa were microinjected into the ooplasm of goat oocytes using a piezo micropipette-driving system (PiezoDrill). In order to assess developmental competence, the ICSI-derived zygotes were cultured in one of two media systems (mTALP-mKSOM vs G1.3-G2.3) for in vitro development or were transferred into recipients for full-term development. The results suggest that cutting sperm tails using the oocyte-holding pipette coupled with the PiezoDrill is an efficient approach for goat ICSI in terms of oocyte survival, pronuclear development and initial cleavage. The mTALP-mKSOM culture system was more suitable for in vitro development of ICSI-derived goat embryos than G1.3-G2.3. This first report of full-term development of an ICSI-derived goat embryo suggests that ICSI can be applied to assisted reproduction in goats.

Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes.

This study was undertaken to investigate various factors affecting the outcomes of in vitro fertilization (IVF) of oocytes retrieved by laparoscopic ovum pick-up (LOPU) technique from prepubertal and adult goats, as well as to evaluate the developmental competence of in vitro produced embryos. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh semen was used for IVF following various capacitation treatments. In vitro produced zygotes were either cultured to assess in vitro development or were transferred into recipients for full term development. The results indicated that successful IVF of the goat oocytes was affected by factors such as sperm capacitation treatment, oocyte quality, and abundance of cumulus cells on zona pellucida. Oocytes from both prepubertal and adult goats demonstrated similar full term developmental competence despite the fact that in vitro developmental rates were lower for prepubertal goats. The births of transgenic offspring demonstrated that the established LOPU-IVF technology combined with pronuclear microinjection can be successfully used to produce transgenic goats.

Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies.

Laparoscopic ovum pick-up (LOPU) is a convenient methodology by which oocytes can be recovered and used either for in vitro production of zygotes or as a source of cytoplasts in nuclear transfer (NT) procedures. The pregnancy and transgenesis rates achieved with IVM/IVF of LOPU-sourced oocytes followed by subsequent DNA microinjection of zygotes are similar to the rates obtained when using in vivo-produced oocytes or zygotes. Similarly, pregnancy rates and kids born by using LOPU-sourced and in vitro matured oocytes as recipient cytoplasts in NT programs are comparable with those reported by others using in vivo matured oocytes collected by oviduct flushing. The use of LOPU allows for improved control over the stage of maturation/development of the oocytes and produced zygotes, a less invasive means of recovery, thereby allowing for repeated usage of the oocyte donor animals and the ability to source the oocytes from live animals of known health status. In addition, because of large follicular responses that can be obtained from prepubertal animals, LOPU followed by IVM/IVF has demonstrated great potential for the early propagation of valuable animals, in particular, transgenic animals.

Production of cloned goats after nuclear transfer using adult somatic cells.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.

Seasonal variation in preovulatory events associated with synchronization of estrus in dwarf goats.

This experiment was conducted to define the temporal relationships among estrus, the LH surge and ovulation after estrus synchronization in dwarf goats and to assess the effect of season on these parameters. In November (breeding season), March (transition period) and July (non-breeding season), estrus was synchronized in 12 dwarf goats by means of intravaginal sponges containing 60 mg medroxyprogesterone acetate (MAP) for 10 d, coupled with 125 microg cloprostenol i.m. 48 h before sponge removal and 300 IU eCG i.m. at sponge removal. A different group of animals was used during each time period. Onset of estrus was monitored using two males, and blood samples for the measurement of plasma LH were collected at 2-h intervals from 24 to 60 h after sponge removal. Ovulation was confirmed by laparoscopy at 54 and 72 h after sponge removal. A seasonal shift was detected in the intervals to onset of estrus, LH surge, and ovulation after sponge removal (P<0.05), with sponge removal to onset of estrus being shorter (P<0.05) in November (25.0 +/- 1.56 h) and July (28.9 +/- 2.43 h) than in March (40.9 +/- 3.27 h). The intervals between onset of estrus and the LH surge and between the LH surge and ovulation were found to be constant throughout the different seasons. An optimal time for breeding, artificial insemination, oocyte and embryo recovery, and embryo transfer may be predicted using information gained from these studies.

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

Sexual maturation and fertility of male Nigerian Dwarf goat (Capra hircus) clones produced by somatic cell nuclear transfer.

Three, genetically identical, Nigerian Dwarf bucks produced by somatic cell nuclear transfer (NT) of fetal fibroblasts were monitored for sexual maturation and fertility. Starting at four months of age, these male clones were trained to serve an artificial vagina (AV). Average age of the NT-derived bucks at first semen collection was 20 weeks, which was not different from that of other young bucks of this breed (average age at first collection = 20 weeks). Average sperm production at 5 months of age for the NT-derived bucks was 5.0 x 10(8) spermatozoa, which was comparable to that of dwarf bucks of similar age (3.4 x 10(8) spermatozoa). At seven months of age, semen collected from two NT-derived bucks was used to artificially inseminate six females (three does per buck). Five does were confirmed pregnant by ultrasound at day 42. Nine healthy kids, four males and five females, were born in March and April 2000. Viable spermatozoa were collected from one of the F1 males at 28 weeks of age. These results demonstrated that NT-derived bucks and one of their male offspring developed sexually within the normal timeframe for their breed and that the clones were fertile.

The storage of pure ram semen at room temperature results in capacitation of a subpopulation of spermatozoa.

We investigated whether storage of pure ram semen at room temperature would facilitate the sperm capacitation process, as assessed by means of the chlortetracycline method. Objective motility, membrane integrity and ability of spermatozoa to undergo acrosome reaction induced by A23187 for 15 min were simultaneously evaluated to gain further insight into this process. Storage for 4 h at room temperature had a clear capacitating effect in approximately 50% of spermatozoa and increased their ability to respond to A23187. Beyond that time, the percentage of motility and membrane integrity remained unchanged. Moreover, storage did not alter the ability of those spermatozoa that remained noncapacitated under these conditions to become capacitated in SOF-m medium. Storage for 4 h increased the percentage of spermatozoa showing swelling of the apical ridge from 3 to 13%. In conclusion, storage of ram semen at room temperature for 4 h in the dark has a marked capacitating effect on a subpopulation of spermatozoa, without changes in motility or membrane integrity, and a low effect on the appearance of the acrosome. Since semen storage is generally included in different IVF protocols, the results presented here contribute toward a clearer understanding of its role in these procedures.

Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining.

We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Ram spermatozoa were treated with fluorescein isothiocyanate-labelled Pisum sativum lectin (PSA), fluorescein isothiocyanate-labelled Arachis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triticum vulgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIC) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC. Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsible for the low fertility of cryopreserved samples.

Changes in sperm-bound amidase activity suggest subtle damage to ram sperm acrosomes by freezing/thawing, not detected by light microscopy.

We have measured sperm-bound amidase activity in fresh, cooled and frozen/thawed ram spermatozoa, in order to study if freezing and thawing led to some degree of acrosome damage of motile/viable spermatozoa not detected by optical methods. This assay was based on the fact that membrane damage would result in an increased access of the enzyme substrate to the sperm acrosome. Semen was collected from adult Australian Merino rams, and spermatozoa were washed by centrifugation through a Ficoll solution. Sperm-bound amidase activity was measured in whole spermatozoa using the protease substrate benzoyl-arginyl-p-nitroanilide (BAPNA). Acrosomal status was also assessed by light microscopy after Giemsa staining. Most amidase activity was shown to be sperm-bound, as only a minor fraction of the enzyme activity was release into the medium after induced damage. Simultaneous assessment of sperm-bound amidase activity and the percentage of spermatozoa with microscopically evident acrosomal damage, after mild sonication for different times, showed a high correlation between both parameters (r = 0.97, p < 0.001). In separate experiments, fresh, cooled and frozen/thawed semen samples were filtered through Sephadex G-10 to obtain a subpopulation of motile, mostly acrosome-intact spermatozoa. As controls, spermatozoa from the same samples to which extensive acrosome damage was induced were evaluated. Slow cooling to 4 degrees C had no effect on amidase activity or percent acrosomal damage with respect to fresh samples. Freezing and thawing resulted in a sperm population that, after filtration through Sephadex, had a low percentage of acrosome damage (9.4%, vs. 2.1% for fresh filtered controls), which was 11% of that obtained after extensive acrosome damage (83%). However, amidase activity in these samples was markedly increased, showing values of activity that were 56% of those obtained in extensively damaged spermatozoa. This effect was not due to an alteration in the enzyme kinetics. We conclude that sperm-bound amidase activity is useful to detect subtle changes, provoked by a standard freezing/thawing procedure, in the permeability of acrosomes from ram spermatozoa which are not detected by direct observation of the acrosomes after Giemsa staining.