Animal Science

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clark garieninus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and ti-eezing in l-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -lO'C/min) to various endpoint temperatures (range -25 to -70°C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (p~O.05) were obtained by spermatozoa frozen at -5"C/min to -45 to -5O'C and at -lO"C/min to -55'C. In 3-step freezing programs, at -5"C/min, the effect of holding spermatozoa for 0,2 or 5 min at -30, -35 or -40°C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P>O.O5) were produced by spermatozoa frozen to, and held at, -35°C for 5 min and at -40°C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5"C/min, 5-min hold at -4O"C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (PXl.05) in hatching rate was observed between frozen and kesh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish. 0 2000 by Elsevier Science Inc.

The aim of this study was to develop a protocol for semen storage of piracanjuba (Brycon orbignyanus) by both cool storage at 4 °C and cryopreservation at − 196 °C. Semen was diluted in some fish semen extenders (Exp. 1) or in extenders combined with the antibiotic gentamycin sulfate (Exp. 2) and stored at 4 °C. Sperm motility was estimated every 24 h. Then, the effects of egg yolk (0 and 5%), cryoprotectants (dimethyl sulphoxide — DMSO, methanol, and methylglycol) and extenders (NaCl 154 mM, BTS™ Minitub and M III™ Minitub) on semen cryopreservation were evaluated (Exp. 3). Semen was added to each of eighteen cryosolutions (2 yolk concentrations × 3 cryoprotectants × 3 extenders), aspirated into 0.5-mL straws, frozen in nitrogen vapor (Taylor-Wharton, CP 300, “dry shipper”) and stored at − 196 °C. Sperm motility was evaluated after thawing at 60 °C-water bath for 8 s. The three cryosolutions that produced the highest post-thaw sperm motility were used again to freeze semen. Post-thaw semen quality was then evaluated under three tests: sperm motility, the percentage of live spermatozoa and hatching rate (Exp. 4). Piracanjuba semen diluted (1:10 total volume) in NaCl 200 mM or in Saad solution (NaCl 200 mM, Tris 30 mM) maintained motility above 35% for as long as 7 days, at 4 °C. Motility of only 7% was observed on undiluted semen after 3 days at 4 °C. There was neither beneficial nor detrimental effect of gentamycin on sperm motility at 250 μg/mL. Egg yolk addition to the cryosolution was beneficial in samples cryopreserved in NaCl 154 mM and in M III™, but detrimental for samples cryopreserved in BTS™. Methylglycol was the most effective cryoprotector compared to DMSO and methanol. Motility and percentage of live spermatozoa were similar among semen cryopreserved in NaCl–yolk, M III™–yolk and BTS™, all containing 10% methylglycol, but lower than fresh control. Hatching rates of eggs fertilized with sperm cryopreserved in NaCl–yolk or BTS™ were higher than for eggs fertilized with sperm cryopreserved in M III™–yolk, but lower than control fertilizations. The semen cryopreservation protocols developed here will be used to set up a gene bank for endangered piracanjuba populations.

In the hatchery-bred African catfish, Clarias gariepinus, spontaneous semen release does not occur and hand-stripping is practically impossible. This reproductive dysfunction may be due to a lack of a pre-spawning gonadotropin (luteinizing hormone—LH) surge. To test this hypothesis, the effects of hormones that increase plasma LH levels were analyzed. Mammalian gonadotropin releasing hormone analogue (mGnRHa), mGnRHa plus pimozide, a dopamine antagonist (mGnRHa-PIM), ovaprim (salmon GnRHa plus domperidone, a dopamine antagonist), carp pituitary suspension (carp-PS), Clarias pituitary suspension and combinations of carp-PS and ovaprim were tested. Stripped fluid, when present, was compared to intratesticular semen, 12 or 24 h after injection (latency time). Plasma LH levels increased (P<0.05) 2 h after injection in all hormone treatments, compared to control fish. Stripping of a few drops of fluid, containing some viable spermatozoa, was possible in four out of five males treated with two injections of carp-PS, sampled 12 h later, and in 13 out of 24 males treated with combinations of carp-PS and ovaprim. Spermatocrit, sperm concentration and hatching rates obtained with stripped fluid, however, were very low compared to those obtained with intratesticular semen, from the same males. The number of sperm cells collected per kg body weight increased only in fish treated with two consecutive injections of carp-PS and a 12-h latency time. Treatments using single injections of pituitary suspensions and treatments with mGnRHa, mGnRHa-PIM or ovaprim did not facilitate hand-stripping of viable sperm cells, nor did they increase the number of sperm cells collected per kg. Based on these results, it is unlikely that hatchery-bred catfish males are not strippable because of a lack of an LH surge.

Cooling and freezing protocols of pirapitinga (Brycon nattereri) semen were evaluated using semen diluted in 154mM NaCl, 200mM NaCl, Saad or BTS™, and cooled for seven days. Sperm motility was daily evaluated. Five extenders (277mM glucose, 154mM NaCl, 200mM NaCl, Saad and BTS™) were combined with two cryoprotectants (DMSO – dimethyl sulphoxide and methylglycol) to produce 10 cryosolutions. Semen was diluted in each cryosolutions, aspirated into 0.5ml straws and frozen. Sperm motility was evaluated after thawing (60°C, 8 sec). Then, semen was frozen in straws with different volumes (0.25 and 0.5ml), and thawed under different water-bath temperatures (50° and 60°C). Higher sperm motility (48%) was observed when semen was cooled in BTS™ for seven days. Post-thawing sperm motility above 68% was observed when semen was frozen in 154mM NaCl-methylglycol, BTS™-methylglycol, 200mM NaCl-DMSO or Saad-DMSO. There was no difference on sperm motility when semen was frozen in 0.25 or 0.5ml straws and thawed in 50° or 60°C water-bath. Thus, pirapitinga semen can be successfully cooled in BTS™ for seven days or frozen in 154 mM NaCl-methylglycol, BTS™- methylglycol, 200mM NaCl-DMSO and Saad-DMSO.

The aim of this study was to test the e¡ects of cryoprotectants [dimethyl sulphoxide (DMSO) and methylglycol], extenders (0.9% NaCl, 5% glucose, Beltsville Thawing Solution TM and Merck III TM ), thawing temperatures (30 and 60 1C) and activating agents (0.29% NaCl and1% NaHCO 3 ) on the cryopreservation process of tiete tetra Brycon insignis sperm. Sperm was loaded in 0.5 mL straws, frozen in nitrogen vapour at À 170 1C and stored in liquid nitrogen. Post-thaw sperm quality was evaluated in terms of subjective motility rate, quality motility score (0 5 no movement; 5 5 rapidly swimming spermatozoa), duration of motility and vitality (eosin^nigrosin staining). Post-thaw sperm motility rate was greater in methylglycol (768 8%), compared with DMSO (23^59%). In general, the highest quality motility scores were observed when sperm was thawed at 30 1C and triggered in 1% NaHCO 3 (3.5^4.3). Duration of motility was longer when triggered in 1% NaHCO 3 (95^120 s) compared with 0.29% NaCl (69^107 s). Sperm vitality was not affected by any of the parameters tested and varied from 51% to 69% intact sperm. Brycon insignis sperm frozen in methylglycol combined with any of the extenders tested and using the methods described above yields motility above 57% and that should last long enough to fertilize oocytes.

The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.

The cryopreservation of fish sperm provides a tool by which reproduction is optimized and thereby larval production is increased. The aims of this study were to evaluate the effects of cryosolutions, motility-activation media, straw volumes and thawing temperatures on the post-thaw motility of curimba semen. Furthermore, semen cryopreserved in a simple and inexpensive cryosolution and that yielded excellent post-thaw motility was tested for fertility. Semen was diluted in each of the eight cryosolutions in a factorial of two cryoprotectants (DMSO and methylglycol) × four extenders (0.9% NaCl, 5% glucose, BTS™ and M III™). Diluted semen was frozen in 0.5-mL straws in a nitrogen vapor vessel. Sperm motility was evaluated after thawing (60 °C water bath for 8 s) and activation with a total of four different activation media (distilled water, 0.15% NaCl, 0.29% NaCl or 1% NaHCO3). To evaluate straw volume and thawing temperature, semen was diluted in 5% glucose and methylglycol and frozen in 0.5- and 4.0-mL straws. Half of the 0.5-mL straws were thawed in a water bath at 60 °C for 8 s and the other half at 30 °C for 16 s. The 4.0-mL straws were thawed at 60 °C for 24 s only. In the last experiment, semen cryopreserved in 5% glucose and methylglycol, 0.5-mL straws, and thawed at 60 °C for 8 s was tested for fertility. The results of these comparisons are presented and show that curimba semen can be successfully cryopreserved in a simple glucose solution combined with methylglycol as cryoprotectant, in 0.5-mL straws, yielding motility rates between 86% and 95% and fertilization rates between 47% and 83%.

The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater ¢sh pirapitinga Brycon nattereri. Extenders (BTS TM and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 1C) and activating agents (0.29% NaCl and 1% NaHCO 3 ) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry-shipper). Post-thawed sperm motility rate, motility quality (score 0 5 no movement; 5 5 rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 mm long, the head was ovoid (2.00 Â 1.22 mm) with no acrosome, the midpiece was 2.15 mm long and the £agellum was 30.90 mm long with the typical 912 axoneme arrangement. Post-thawed sperm motility rate (70^79% motile sperm), motility quality (score 3.1^3.7) and morphology (9.3^11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO 3 (392^1031s) compared with 0.29% NaCl (144^338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.

The effects of 17α-methyltestosterone on seminal vesicle development in the African catfish, Clarias gariepinus, were investigated in an attempt to improve semen collection from this species. Treatment of larvae with dietary 17αmethyltestosterone at 50 mg kg -1 for days 12-33 or days 12-40 after hatching, or at 20 mg kg -1 for days 12-26, 12-33, 12-40 or 12-47 after hatching inhibited the development of the seminal vesicle finger-like extensions in male catfish, but did not affect the sex ratio. The minimum effective dose and period of treatment to inhibit seminal vesicle development in all male catfish treated with 17αmethyltestosterone was 20 mg kg -1 for days 12-40 after hatching. Male catfish from this treatment group developed normal testes that, in some cases, contained a few oocytes, which tended to disappear before sexual maturation. After sexual maturation, the semen release response was evaluated in males with incomplete seminal vesicles. Fluid with viable spermatozoa was obtained after two consecutive injections of carp pituitary suspension, from 10 of 19 males that had been fed 20 mg 17α-methyltestosterone kg -1 for days 12-40 or days 12-47 after hatching, but from only 4 of 15 males that did not receive any dietary steroid. Intratesticular semen quality was not affected by 17αmethyltestosterone treatment. The results of this study demonstrate that the absence of seminal vesicle extensions induced by treatment with 17α-methyltestosterone facilitated the collection of semen by stripping from this species of fish.

The aims of this study were to evaluate the e⁄ciency of simple and complex extenders in prolonging the cold storage of sperm (Experiments 1 and 2) and to test the diluted-cooled sperm in the best extender with regard to sperm quality parameters (Experiment 3) in the streaked prochilod, Prochilodus lineatus. In all the experiments, aliquots of 0.3 mL of sperm were diluted 1:10 in extenders and stored at 4^6 1C. Sperm diluted in simple extenders (NaCl and glucose solutions) yielded 0^26% sperm motility, whereas sperm diluted in complex extenders (BTS TM , M III TM and Androstar TM ) yielded 62^81% sperm motility on day 4 after cold storage. When Androstar TM was further investigated, the following was observed on day 4: 53% motility with 94 s of duration; 47% live spermatozoa; 26^61% fertility rate; and 22^60% hatching rate. The use of Androstar TM improves the sperm fertility of the streaked prochilod during a 4-day storage period and can therefore be used to facilitate arti¢cial reproduction.

In silurid fishes, semen collection is practically impossible, even after hormonal stimulation. Instead, males are killed and testes macerated to obtain sperm. To understand the endocrine control of semen release in catfishes, we investigated the role of smooth muscle contractors in semen release and semen quality of African catfish (Clarias gariepinus). In in vitro experiments, testis slices were incubated with oxytocin (1 and 10 IU), isotocin (2 and 20 ug), vasopressin (0.2 and 2 ug), epinephrine (1 and 10 ug), PGF2α (1 and 10 ug), purified Clarias LH (300 ng) and partly purified Clarias pituitary extract (containing 300 ng LH). Only oxytocin increased sperm concentration of the medium (assessed by optical density measurements) compared to control incubations. Oxytocin was then tested in vivo in two groups of fish: normal males, and males that had been treated with 17α-methyltestosterone during larval stages to inhibit seminal vesicle development (MT males). Both groups of fish received two doses of carp pituitary suspension (8 and 10 mg/kg, respectively i.m.) with or without subsequent oxytocin treatment (5 IU/kg i.v.; cPS-OT treatment and cPS treatment, respectively). There was no effect of oxytocin on the number of strippable males. Of cPS and cPS-OT treated fish, 87% MT males and 60% normal males were strippable. The stripped semen volume was low in both groups but MT males produced higher (P<0.001) hatching rates (63.1%) than did normal males (2.1%).

The objective of this study was to optimize interrupted slow-freezing protocols for African catfish semen. Semen diluted with methanol and extender was frozen in 1-ml vials in a programmable freezer. The temperatures of the freezer (Tchamber) and of the semen (Tsemen) were measured simultaneously. We first tested two-step freezing protocols with different cooling rates (−2, −5, and −10°C/min) and different temperatures at plunging into liquid N2. The difference between Tsemen and Tchamber increased with faster cooling rates. In all programs, survival of spermatozoa, expressed as hatching rates, increased from near zero when Tsemen at plunging was higher than −30°C to values equal to those of control when Tsemen at plunging was equal to or lower than −38°C. The inclusion of an isothermal holding period before plunging into liquid N2 (three-step freezing protocols) resulted in an equilibration between Tsemen and Tchamber and improved semen survival. Semen could be plunged at temperatures as high as −36°C when cooled at −5 or −10°C/min, without compromising postthaw semen survival. Cooling at −2°C/min in combination with a 5-min holding period reduced postthaw survival. We conclude that with slow cooling rates of −2 to −5°C/min, hatching rates can be maximized by plunging as soon as Tsemen reaches −38°C. The isothermal holding period is beneficial when faster rates are used. A simple and efficient protocol for freezing African catfish semen can be obtained by cooling at a rate of −5 to −10°C/min combined with a 5-min holding period in the freezer, at −40°C

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 mm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 mm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg À1 (average: 283.88 AE 33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P < 0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg À1 . Osmolality above 375 mOsmol kg À1 inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters. Crown

The effects of extenders and cryoprotectants on sperm motility of the endangered fish piracanjuba (Brycon orbignyanus, Characiformes, Characidae) after storage at 4-6ºC and at -196ºC were evaluated. In Experiment 1, 20 extender-cryoprotectant combinations (5 extenders x 3 cryoprotectants, plus 5 solutions containing only extender without cryoprotectant) were tested. The extenders tested were: 154 mM NaCl, 200 mM NaCl, Saad (mM: 200 NaCl, 30 Tris), coconut water, and Kurokura (mM: 128.4 NaCl; 2.7 KCl; 1.4 CaCl 2 ; 2.4 NaHCO 3 ). The cryoprotectants (dimethyl sulphoxide -DMSO, methanol, and methylglycol) were added at 10% of the total volume. One aliquot of semen was kept undiluted and served as a control. Motility was subjectively estimated at 0, 1, and 2 days after cold storage. In Experiment 2, the extender-cryoprotectant combinations that produced motility above 70% on Day 0 (Experiment 1) were selected as freezing media. The amount of egg yolk and semen included in each medium was 5 and 10% of the total volume, respectively. Three 0.5-mL straws for each freezing medium were frozen in nitrogen vapor container (Taylor-Wharton, CP 300) at -170°C and then stored in liquid nitrogen. Straws were thawed in a water bath at 60ºC for 8 seconds. Sperm motility one day after cooling was higher in samples diluted in Saad solution (82%), 200 mM NaCl (67%), and 154 mM NaCl -DMSO (77%). Undiluted samples yielded 53% motility. Higher post-thaw sperm motility (66%) was observed in semen cryopreserved in 154 mM NaCl -egg yolk -methylglycol compared to all the other samples. Piracanjuba semen diluted in Saad solution or 200 mM NaCl and stored at 4-6ºC for one day or frozen in 154 mM NaCl -yolk -methylglycol maintained most of its sperm motility.

The pirapitinga (Piaractus brachypomus) is a freshwater fish that inhabits the Amazon and Orinoco River basins. The use of cryopreserved sperm has been considered to facilitate procedures during the artificial reproduction. The aim of the present study was to develop a freezing protocol for pirapitinga sperm collected outside the spawning season. Sperm samples were diluted in four freezing media prepared by a combination of two extenders (glucose and BTS™—Beltsville Thawing Solution) and two cryoprotectant agents (DMSO and methylglycol) loaded into 0.5-mL straws, frozen in a nitrogen-vapor shipping dewar (dry-shipper) and stored in liquid nitrogen at −196 °C. Post-thaw sperm motility was evaluated both subjectively using a light microscope and by a computer-assisted sperm analyzer (CASA). Curvilinear, average path and straight-line velocities were also determined. There were no differences (P > 0.05) in post-thaw sperm motility between evaluations performed subjectively and using the CASA. Sperm samples cryopreserved in glucose-methylglycol yielded the greatest post-thaw sperm motility (81%) and fastest sperm velocities when compared to the samples frozen in the other three media (P < 0.05). Out-of-season sperm cryopreserved in glucose and methylglycol under the conditions described above is of high quality and can therefore be used to facilitate artificial reproduction procedures, as only females will need handling for hormonal induction and gamete collection during the spawning season. Although the CASA system provides precise data on sperm motility, the subjective evaluation is practical and can be conducted by well-trained personnel at commercial fish farms as an acceptable evaluation of sperm quality.

Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP™), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 °C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP™ (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46–48%) and sperm velocities. There were positive correlations (r = 0.56–0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP™ and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.

Sperm preservation is an important tool for conservation of endangered fish species, such as the Brycon opalinus (Characiformes). An optimum medium should prevent the initiation of sperm motility during storage. The aim of this paper was to study the effects of extender composition, osmolality and cryoprotectant agent (CPA) on the motility of B. opalinus sperm after a 30-min equilibration time. Eight media were prepared by switching extender compositions (NaCl or glucose) and osmolalities (245, 285, 325 or 365 mOsm/kg). These media were then divided in three aliquots and combined with two CPAs (dimethyl sulfoxide, DMSO, or methylglycol, MG) at 1.4 M; the third aliquot remained without CPA (control). After dilution, all samples were observed under light microscope to confirm whether all extender-CPA combinations actually prevented the initiation of sperm motility. Then, sperm motility was triggered in NaCl 92 mOsm/kg as activating agent after 0- and 30-min equilibration time at 4 °C and the percentage of motile sperm and duration of motility were determined. All combinations of glucose or NaCl media at high osmolalities (325 and 365 mOsm/kg), completely suppressed the initiation of sperm motility. Low extender osmolalities (245 mOsm/kg), however, did not prevent the initiation of sperm motility and more than 50% of sperm diluted in all combinations of glucose media, NaCl-control and in NaCl-DMSO were motile. When motility was triggered after both 0- and 30-min equilibration times, more than 77% motile sperm were observed in all combinations of NaCl and glucose media, except for glucose 245-DMSO and glucose 285-DMSO. The duration of motility in sperm diluted in all media was above 40 s, except for glucose 245-DMSO. Based on these findings, we can conclude that the initiation of sperm motility is triggered by low osmolality rather than the ionic composition of the surrounding medium in B. opalinus. Glucose or NaCl solutions at high osmolalities combined with either DMSO or MG prevent the initiation of sperm motility during storage. Sperm diluted in these media yields motility upon activation above 77% and that should last long enough to fertilize oocytes. These media are recommended as freezing media for future essays in cryopreservation of B. opalinus sperm.

The objective was to evaluate the effect of four different dosages of retinol palmitate 0 - (n=14), 500,000 (n=15), 1,000,000 (n=17) and 1,500,000 (n=16) International Units (IU) of vitamin A in the form of retinol palmitate on production and quality of embryos recovered from Nelore donor cows (n=64). The experiment was carried out in the Embryo Transfer Company Cauembryo in the county of Funilândia - MG. Cows were superovulated on the 10th (n=18),11th (n=10),12th (n=26) or 13th (n=10) days after estrus onset with an injection of 20 ml of Folltropin® (Vetrepharm, Belleville, Canada) or 10 ml of Pluset® (I.F. Serono,Roma, Itália) administered in decreasing doses for four days twice daily (each 12 h). Vitamin A injection was delivered with the first FSH injection. Luteolysis was induced on the 4th day of FSH treatment by giving 0.75 mg of sodium cloprostenol (Ciosin®, Coopers do Brasil, São Paulo, Brasil) and donors observed in estrus were artificially inseminated 12 and 24 hours after the onset of estrus using semen of different bulls of proven fertility. Statistical analysis was carried out using the GENMOD procedure of SAS (SAS INSTITUTE, 1995). The number of viable embryos differed (P<0.0001), between control (3.6), and 500,000 (6.1), 1,000,000 (6.5) and 1,500,000 (6.7) vitamin A treatments. Percentage of viable embryos also increased (P<0.01), from 0.51 in the control, to 0.57, 0.63 and 0.60 in order of increasing Vitamin A treatment groups, respectively. Number of total structures recovered (ER) was not different between supplemented (9.5, 8.3 and 10.5 respectively for 500,000, 1,000,000 e 1,500,000 IU of vitamin A) and not supplemented (8.2). These results indicate that supplementation of vitamin A i.m. before flushing improves embryo quality without interfere in the quantity of embryos recovered.

The objective was to evaluate the effect of four different dosages of retinol palmitate 0 - (n=14), 500,000 (n=15), 1,000,000 (n=17) and 1,500,000 (n=16) International Units (IU) of vitamin A in the form of retinol palmitate on production and quality of embryos recovered from Nelore donor cows (n=64). The experiment was carried out in the Embryo Transfer Company Cauembryo in the county of Funilândia - MG. Cows were superovulated on the 10th (n=18),11th (n=10),12th (n=26) or 13th (n=10) days after estrus onset with an injection of 20 ml of Folltropin® (Vetrepharm, Belleville, Canada) or 10 ml of Pluset® (I.F. Serono,Roma, Itália) administered in decreasing doses for four days twice daily (each 12 h). Vitamin A injection was delivered with the first FSH injection. Luteolysis was induced on the 4th day of FSH treatment by giving 0.75 mg of sodium cloprostenol (Ciosin®, Coopers do Brasil, São Paulo, Brasil) and donors observed in estrus were artificially inseminated 12 and 24 hours after the onset of estrus using semen of different bulls of proven fertility. Statistical analysis was carried out using the GENMOD procedure of SAS (SAS INSTITUTE, 1995). The number of viable embryos differed (P<0.0001), between control (3.6), and 500,000 (6.1), 1,000,000 (6.5) and 1,500,000 (6.7) vitamin A treatments. Percentage of viable embryos also increased (P<0.01), from 0.51 in the control, to 0.57, 0.63 and 0.60 in order of increasing Vitamin A treatment groups, respectively. Number of total structures recovered (ER) was not different between supplemented (9.5, 8.3 and 10.5 respectively for 500,000, 1,000,000 e 1,500,000 IU of vitamin A) and not supplemented (8.2). These results indicate that supplementation of vitamin A i.m. before flushing improves embryo quality without interfere in the quantity of embryos recovered.