Transferable embryo recovery rates following different insemination schedules in superovulated beef cattle

The objective of this study was to evaluate the transferable embryo recovery rates from superovulated donor cattle after different artificial insemination (AI) schedules. Sixty mixed-breed crossbred females were administered follicle stimulating hormone (FSH) and prostaglandin F(2)alpha (PGF(2)alpha) to induce a superovulatory response. At standing estrus, donor females were randomly allotted to one of five treatment groups for AI. Donors were inseminated with two units of high-quality or low-quality frozen semen at 12, 24, 36, or 48 h after the onset of estrus in treatment Groups I, II, III, and IV, respectively, or inseminated with two units at 12, 24, 36, and 48 h (eight units/donor) in control Group V. Donor females inseminated once at either 12 or 24 h after the onset of estrus did not differ from donors inseminated in Group V in overall fertilization and transferable embryo recovery rates. The highest fertilization rate (89.5%) and transferable embryo recovery rate (74.9%) per donor resulted when AI was performed with high-quality semen at 24 h after the onset of estrus. These findings indicate that repeated insemination of superovulated beef cattle is not necessary to attain optimal fertilization rates and production of transferable quality embryos in beef cattle.     

Embryo quality and andrological study of two subfertile bulls versus five control bulls with normal fertility

Andrological studies and embryo morphology evaluation of superovulated cows were performed on 2 randomly selected subfertile dairy bulls whose semen was used for artificial insemination and on 5 control bulls with normal fertility. Neither sperm motility studies, nor sperm morphology or testicular measurements differed between the subfertile and the control bulls. Altogether 315 ova were recovered from 41 superovulated cows inseminated with semen collected from either the subfertile or the normal control bulls. The spermatozoa of one of the 2 subfertile bulls was shown to have a decreased ability to fertilize superovulated ova, while the other subfertile animal, the bull with the lowest noreturn rate, was found by chromosome analysis to have a reciprocal translocation (60, XY, rcp 20:24), causing embryonic death. We suggest that subfertile bulls should not be used in commercial embryo transfer programs nor in artificial insemination and that andrological studies on subfertile bulls with good sperm motility should include evaluation of 6- to 7-day-old ova from superovulated cows to determine if the fertilization rate is normal or impaired. A chromosome analysis should also be performed when a subjertile bull has a normal fertilization rate of ova.     

Determination of the fertilization rate of A.I. sires

The ovarian cycles of 160 beef heifers were controlled with two injections of PGF_2α and superovulation was induced by 2000 IU PMSG, administered 48 h prior to the second PGF_2α treatment. Heifers were inseminated with fresh or frozen semen from selected bulls that demonstrated consistent NR's above (n=5) or below (n=3) the stud average. Sixty-five percent of ovulated ova were recovered from the responding heifers. The mean fertilization rates for the above and below average fertility groups in this study were 89.0% (355 ova) and 69.7% (155 ova) respectively. This pilot study indicated the possibility of detecting differences in the fertilization rate between bulls using superovulated females to obtain large quantities of ova from relatively few experimental animals. Further research with more bulls would be required to accurately determine the correlation between fertilization rate and nonreturn rate but the results suggest the procedure may have application for selection of potential AI sires with above average fertility.     

Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen

Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.     

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis)

The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.     

Semen and reproductive profiles of genetically identical cloned bulls

In this comparative study, reproductive parameters and semen characteristics of cloned bulls (n = 3) derived from somatic cell nuclear transfer (SCNT) were compared to their original cell donor Holstein-Friesian (n = 2) bulls from the same enterprise to assess the differences in reproductive potential between a donor bull and its clones. The parameters evaluated included motility of fresh, frozen-thawed and Percoll-treated frozen-thawed spermatozoa, as well as in vitro fertilization (IVF) ability, embryo quality, birth and survival of calves following IVF and embryo transfer with frozen-thawed semen. With fresh semen, spermatozoa from one cloned bull had lower motility than its donor. Cloned bulls had higher velocity parameters in fresh semen, but those effects were not obvious in frozen-thawed or frozen-thawed semen selected with a Percoll gradient. Semen collected from cloned bulls had significantly higher IVF rates compared to donors; however, embryo development per cleaved embryo or quality of blastocysts did not differ between donors and cloned bulls. Pregnancy and live offspring rates from one donor and its cloned bull did not differ between fresh (40%, 16/40 versus 46%, 17/37) and vitrified/thawed (13%, 2/16 versus 25%, 4/16) embryo transfer following IVF. A total of 26 calves were obtained from genotypically identical donor and cloned bulls with no signs of phenotypical abnormalities. These preliminary results suggested that the physiology of surviving postpubertal cloned bulls and quality of collected semen had equivalent reproductive potential to their original cell donor, with no evidence of any deleterious effects in their progeny.     

Superovulatory response, embryo quality and fertility after treatment with different gonadotrophins in native cattle

We studied native Mertolengo cattle to evaluate superovulatory (SOV) treatments, subsequent fertility of donors and pregnancy rate of recovered embryos. In Experiment 1 we compared superovulatory response (SR), embryo quality and plasma progesterone (P4) levels between donors treated with eCG (10 cows and 5 heifers) vs. FSH (pure, FSH-1, n=10 cows and crude, FSH-2, n=10 cows), during progestagenic impregnation. We also compared fertilization rates and embryo quality of bred and inseminated eCG and FSH-1 donors. Significantly more viable embryos were yielded by FSH than by eCG treated donors. Less FSH-1 than FSH-2-treated donors showed SR, but the response was identical in responder donors of both groups. Fertilization rates were significantly higher in bred than in inseminated donors. Plasma P4 levels were only significantly different (higher) between responder and non-responder donors on the day of embryo recovery. Experiment 2 compared FSH treatments (FSH-2, crude, n=11 cows and FSH-3, pure, n=10 cows) started at the midluteal phase. The mean number of viable embryos was significantly higher in FSH-3 than in FSH-2 treated donors. Both FSH treatments exerted a similar luteotrophic effect upon injection. The FSH-2 donors treated during the midluteal phase yielded more ova and showed significantly higher plasma P4 levels at all sampling days than those treated during progestagenic impregnation. The pregnancy rates of recipient cows were 67% and 46% for fresh and frozen-thawed embryos respectively. In Experiment 3, the fertility of donors (n=20) after SOV treatments was compared with that of untreated cows (n=40). Time to conception of donors, after mating with a bull 14 days after embryo recovery, was identical to that of control cows. There was some delay to conception in eCG-treated cows, but the difference was not significant. These preliminary results suggest that response to SOV treatments in Mertolengo cattle might be affected by the type of gonadotrophin and by the treatment protocol. The fertility of a traditional breeding season after SOV treatments was not impaired. Cryopreserved embryo banking can be used to preserve the breed.     

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.     

Evaluation of fertility status of Murrah buffalo bulls in vitro using zona-free hamster eggs

Cryopreserved semen samples from 10 Murrah buffalo bulls were used for sperm penetration bioassay using zona-free hamster oocytes. The samples were evaluated for sperm motility, viability and acrosome integrity. Actively motile spermatozoa recovered by the swim-up technique were capacitated using calcium ionophore A(2 3 1 8 7). Mature female golden hamsters were superovulated with 50 IU PMSG followed 56 h later by 75 IU hCG. Cumulus mass, recovered by puncture of oviducts at the infundibulum region, was treated with 0.1% hyaluronidase and 0.1% trypsin to obtain zona-free oocytes. After coincubation of zona-free oocytes with capacitated buffalo spermatozoa, scoring was done as fertilization percentage and fertilization index. The correlation coefficients with conception rate were statistically significant with fertilization percentage (r = 0.588, P < 0.05) and fertilization index (r = 0.660, P < 0.01). However, conventional parameters like viability, motility and acrosome integrity showed poor correlation with conception rate.     

Does the 1/29 Robertsonian translocation affect the fertility of Blonde d'Aquitaine breed bulls?

The effects of the 1 29 translocation upon male fertility were studied by analysis of the results of 1 350 385 first artificial inseminations with the semen of Blonde d'Aquitaine or Coopelso-93 bulls (n=220). A binomial logit model was designed, taking into account the translocation of sire, breed of sire, breed of dam, year, AI center, and all interactions between translocation, and breed of sire, and breed of dam. Male fertility was not affected by the 1 29 translocation, and the nonreturn rates at 60 to 90 days of Blonde d'Aquitaine females inseminated with the semen of carrier bulls (135 632 first AI) or noncarrier bulls (585 949 first AI) were 74.88% and 74.75%, respectively.     

Factors affecting the yield of viable embryos by superovulated Holstein-Friesian cows

GnRH treatment (250 ug) 48 h after prostaglandin F(2alpha) in 40 superovulated cows induced a release of LH (increment > 5 ng/ml) in only 13 of the older cows. Eleven of these cows did not yield viable embryos. Thirty-two of 75 cows had preovulatory surge levels of LH 48 h after prostaglandin treatment. Plasma progesterone concentrations were determined in 140 cows at the time that superovulation was initiated. Eighty-four of these donors were superovulated with 40 mg of FSH and 56 donors with 48 mg of FSH. There was no relationship (P > 0.05) between the concentration of progesterone at the start of superovulation with either ovulation rate determined by palpation per rectum or the number of viable embryos per flush. These parameters were also unaffected (P > 0.05) by age of the donor or the dose of FSH. In another group of donors, treatment with 40 mg FSH was compared over a 3-d (n = 28) and a 4-d (n = 18) interval. The donors treated with FSH over a 3-d period had similar ovulation rates but yielded less viable embryos (1.5 v 5.8, P < 0.05). The fertility rate of 33 cows, inseminated 60 and 72 h after prostaglandin, was comparable to the fertility rate of 18 cows inseminated at 60, 72 and 84 h after prostaglandin treatment.     

Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida

The objective was to compare conception rates to embryo transfer relative to AI, during summer heat stress, in lactating dairy cows. Holstein cows (n = 180; 50 to 120 d postpartum) were allocated randomly to 1 of 3 groups: artificial insemination (AI, n = 84), embryo transfer using either embryos collected from superovulated donors (ET-DON, n = 48), or embryos produced in vitro (ET-IVF, n = 48). Embryos from superovulated donors were frozen in 10% glycerol and were rehydrated in a 3-step procedure, in decreasing concentrations of glycerol in a sucrose medium before transfer. Embryos produced in vitro were frozen in 1.5 M ethylene glycol, thawed and transferred without rehydration. Blood samples were collected from AI and ET recipients on Days 0, 7 and 22 for measurement of progesterone in plasma. Conception rate was estimated for the three groups at Day 22 (progesterone > 1 ng/mL) and confirmed at Day 42 by palpation per rectum. Conception rate estimates at Day 22 did not differ among groups (AI, 60.7%; ET-DON, 60.4%; ET-IVF, 54.2%), but conception rates at Day 42 differed (AI, 21.4%; ET-DON, 35.4%; ET-IVF, 18.8%; AI versus ET: P > 0.10 and ET-DON versus ET-IVF: P < 0.05). In cows considered pregnant at 22 d but diagnosed open at 42 d, the interestrous intervals were 28.8 +/- 2.2, 35.2 +/- 3.5 and 31.6 +/- 2.9 d, respectively, for AI, ET-DON and ET-IVF groups. Transfer of embryos collected from nonheat-stressed superovulated donors significantly increased conception rates in heat stressed dairy cattle. However, transfer of IVF-derived embryos had no advantage over AI. Where appropriate mechanisms are in place to attenuate the effects of heat stress, embryo transfer using frozen-thawed donor embryos increases conception rates.     

Individual differences in capacitation of bull spermatozoa by heparin in vitro: relationship to fertility

Several parameters of motility and integrity of frozenthawed spermatozoa are compared with the ability of selected motile, intact spermatozoa to acrosome reaction induced by 0.005% hyamin. Between semen donors there exist distinct individual differences; however only the induced acrosome reaction after heparin treatment showed a significant correlation with fertility. For 12 bulls the nonreturn rates from 334 to 559 services correlated significantly with induced acrosome reaction (r = 0.607). The in vitro fertilization of 53 to 93 tubal bovine oocytes with frozen-thawed spermatozoa from five bulls yielded a correlation of r = 0.621 with the rate of induced acrosome reaction. The different capacity levels of heparin-treated spermatozoa to undergo acrosome reaction appears to correspond to the varying intensity and kinetics of heparinmediated head-to-head aggregation of motile cells. The applied functional parameters could be used for the selection of bulls with low fertility in artificial insemination programs, and for spermatozoa donors for in vitro fertilization.     

Rate of transport and development of preimplantation embryo in the superovulated buffalo (Bubalus bubalis)

This study was designed to ascertain the rate of transport and development of preimplantation embryo in the superovulated buffalo in order to determine the optimum time for their nonsurgical collection. Eighteen Murrah-type buffalo were superovulated with 600 mg NIH-FSH-P1. Luteolysis was induced by administration of PGF2 alpha at 72 (PG + 72) and 84 h (PG + 84) after initiating gonadotrophin treatment and fixed-time AI was done beginning at 36 h post PG + 72 administration and at 12-h intervals thereafter, upto 72 h. Six control buffalo received treatment similar to experimental group except that in place of FSH they received normal saline. For embryo collection, experimental animals were humanely killed at 6-h intervals corresponding to 156 (n = 2), 162 (n = 2), 168 (n = 2), 174 (n = 3), 180 (n = 3), 186 (n = 3) and 192 h(n = 3) after PG + 72 treatment, whereas the control animals were humanely killed at 156 (n = 2), 174 (n = 2) and 192 h (n = 2). Superovulated buffalo had higher number of ovulations than untreated controls (8.78 +/- 5.00 vs 0.67 +/- 0.51) and total ova/embryos recovered was 4.11 +/- 2.46 and 0.67 +/- 0.51, respectively. The high estradiol-17 beta (E2) levels with its prolonged rise may, by leading to reverse peristalsis in the oviduct with a consequent loss of some embryos in the peritoneal cavity, be one of the reasons for our inability to recover nearly 84/158 ova/embryos in the superovulated buffalo. In superovulated animals, nearly all the ova/embryos reached the uterus between 168 and 174 h post PG + 72 treatment or about 134 h (circa 5.5 d) after the onset of superovulatory estrus, suggesting that the ideal time for non-surgical embryo collection in the buffalo is between Days 7 to 8 after PG + 72 treatment or Days 5.5 to 6.0 of the superovulated cycle (estrus = Day 0). Embryo development of superovulated buffalo showed considerable variation as various stages of embryos (8 cell to expanded blastocyst) were recovered from the same donor buffalo, and the rate of development appeared to be 24 to 36 h faster than in cattle.     

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.     

Relationship between bull field fertility and in vitro embryo production using sperm preparation methods with and without somatic cell co-culture

Experiments were designed to compare rates of embryonic development following oocyte exposure to cryopreserved spermatozoa from bulls of varying proven fertility, utilizing 3 different sperm preparation methods prior to oocyte introduction. These included 1) sperm co-culture with bovine oviductal epithelial cells (BOEC); 2) sperm co-culture with buffalo rat liver cells (BRLC); or 3) control culture in a routine, cell-free culture system. Semen from 9 bulls was classified by lifetime 60- to 90-d nonreturn rates as having either (mean +/- SEM) high (n=3) 73.2 +/- 3a, medium (n=3) 70.3 +/- 2b or low (n=3) 65.8 +/- 3c field fertility ((ac)p< 0.01; (bc)p< 0.05). There was no difference in embryo cleavage rates for spermatozoa from the high (58 +/- 18%), medium (57 +/-23%) or low (57 +/- 18%) fertility groups. Development to morula or beyond of oocytes fertilized with high (53 +/- 30%) or low (58 +/- 27%) fertility semen tended (P<0.10) to be higher than of those fertilized with medium fertility (33 +/- 28%) semen. This lack of relationship between in vivo fertility and in vitro embryo outcome was consistent across all sperm preparation methods. Therefore, pooled data were used to evaluate the effect of sperm preparation on embryo outcome. There was no difference in embryo cleavage rates between BOEC monolayers (51 +/- 22%), BRLC monolayers (60 +/- 20%) and the cell-free controls (60 +/- 17%). Subsequent embryonic development to compact morula and beyond was higher (P<0.01) with the BRLC monolayer treatment (61 +/- 28%) than with the BOEC monolayers (42 +/- 33%) or control culture (39 +/- 24%). In conclusion, these studies suggest that there is no predictive relationship between bull field fertility (in the ranges evaluated here) and in vitro embryo cleavage or development rates. However, oocytes inseminated with sperm cells co-cultured on BRLC monolayers develop to the morula stage or beyond at a higher rate than oocytes inseminated with spermatozoa from the BOEC or cell-free system.     

Development of a new transcervical artificial insemination method for sheep: effects of a new transcervical artificial insemination catheter and traversing the cervix on semen quality and fertility

The difficulty of traversing the cervix severely limits transcervical artificial insemination (TC AI) in sheep. Cervical trauma and poorly designed instruments can reduce fertility after AI. To overcome problems associated with TC AI, we developed a new TC AI catheter. Three bench experiments were conducted to determine the effects of the new TC AI catheter on semen quality independent of the effects of moving the catheter through the cervix. In each of the three bench experiments, the standard laparoscopic instrument for intrauterine AI in sheep was used as the control for the TC AI catheter. In Experiment 1, the total volume of semen extender expelled and void volumes for both types of AI instrument (TC versus laparoscopic) were determined. In Experiment 2, the effects of each type of AI instrument (TC versus laparoscopic) on semen quality, estimated as percentage motility and percentage forward progressive motility, of frozen-thawed semen was determined. In Experiment 3, the effects of both types of AI instrument (TC versus laparoscopic) on number of spermatozoa expelled was determined. The type of AI instrument affected neither semen quality nor the number of spermatozoa expelled. However, void volume differed (P < 0.01) between the two instruments. After differences in void volume were taken into account, an in vivo experiment was conducted to determine whether using our new TC AI catheter for TC or surgical intrauterine AI affected fertilization and pregnancy rates. For this, ewes were assigned to one of three treatments: (1) TC AI using the new TC AI catheter + sham AI via laparotomy (n = 9); (2) sham TC AI + AI via laparotomy using a laparoscopic AI instrument (n = 8); and (3) sham TC AI + AI via laparotomy using the new TC Al catheter (n = 10). To synchronize estrus, progestogenated pessaries were inserted and left in place for 12 days. On Day 5 after pessary insertion, PGF2alpha (15 mg) was given i.m. At pessary removal, 400 IU of eCG were administered i.m. Ewes were inseminated 48-52 h after pessary removal using fresh diluted semen (200 x 10(6) to 350 x 10(6) spermatozoa per 0.2 ml) pooled from the same four rams each day during the experiment. At 72 h after AI, uteri were collected postmortem and flushed. Oocytes and embryos were recovered and evaluated. Treatments did not affect (P > 0.01) ovum and embryo recovery rate (mean = 87.3%), fertilization rate (59.3%), or Day 3 pregnancy rate (mean = 66.6%). We conclude from these data that the use of our new TC AI catheter for TC AI or intrauterine AI should not impair the success of AI in sheep.     

Early oocyte penetration can predict the efficiency of bovine embryo production in vitro

The aim of this work was to characterize oocyte fertilization and embryo cleavage in nine AI bulls to find parameters suitable for prediction of in vitro fertility. According to the d8 blastocysts rate, they were categorized as high, medium and low productive (HP, MP and LP, mean: 25.4, 21.0 and 13.6% respectively) bulls. For these categories, oocyte penetration and fertilization efficiency were assessed at 6 and 18 hours post insemination (hpi), respectively. Some presumptive zygotes were cultured and cleaved and fast-cleaved embryo rates were checked at 44 hpi. The penetration rate was significantly higher for HP bulls than for MP and LP bulls (67.9 versus 50.3 and 33.1%; p<0.01). The syngamy rate was significantly higher for HP bulls than for MP and LP bulls (21.4 versus 10.2 and 5.7%; p<0.05). Conversely, no significant differences in fertilization rates were found among HP, MP and LP bulls. The cleavage rate was significantly higher for HP than LP bulls (82.4 versus 74.4%; p<0.01). The fast cleavage rate was significantly higher for both HP and MP bulls, as compared with LP bulls (82.1 and 84.7 versus 73.5%; p<0.01). A strong correlation was found between blastocyst production and penetration (r=0.803), syngamy (r=0.826), cleavage (r=0.635) and fast cleavage (r=0.709). In conclusion, all the evaluated parameters showed a predictive value, the most significant being early penetration and syngamy.     

Testicular development and its relationship to semen production in Murrah buffalo bulls

The objectives of this study were to determine the relationship of age and body weight to testicular development and to establish norms for breeding soundness evaluations of Murrah buffalo bulls. Testicular measurements of 133 Murrah buffalo bulls of various ages were recorded with a caliper and a tape. Semen was collected twice a week for 5 weeks from groups of bulls which were 25-36 (n=17), 37-48 (n=16), 49-60 (n=14), of >60 (n=10) months of age. After examining volume, sperm concentration, and progressive motility semen was diluted in Tris-citric acid-egg yolk-fructose extender and frozen in 0.5 ml French straws. Testicular measurements of buffalo bulls were lower than those recorded for European breeds of cattle bulls. Nevertheless, like cattle bulls, scrotal circumference was highly correlated with other testicular measurements. Also, it had a significant positive relationship with semen volume and sperm concentration per ejaculate. Average sperm output per week in order of increasing age group was 15.3, 18.2, 19.8 and 23.6 x 10(9). Corresponding values for sperm output per week per gram of testis were 59.1, 45.8, 41.1, 36.2 x 10(6) indicating a reduction in spermatogenesis per unit of testis with advancing age. Compared to European breeds, daily sperm output in Murrah bulls was nearly 45% lower, presumably due to their nearly 40% lower scrotal circumference than Holstein bulls of the same age. These results indicate that in buffalo, as in cattle, scrotal circumference is a useful indicator of potential sperm output and may serve as an important criterion for selecting young bulls as AI sires.     

Relationship between embryo development in vitro and 56-day nonreturn rates of cows inseminated with frozen-thawed semen from dairy bulls

Frozen-thawed bull semen with > 50% post-thaw motility from 40 batches (21 bulls, 2 consecutive ejaculates per batch) was used for fertilization (IVF) and embryo development in vitro to assess the relationship between field and laboratory fertility using a retrospective approach. Each frozen batch was tested in 3 or 4 replicates with 30 oocytes per replicate. Field fertility, quantified as the 56-d nonreturn rate and based on 89 to 441 artificial inseminations per frozen batch, ranged between 46.2 and 74.8%. The cleavage and blastocyst rates after IVF varied from 29.0 to 81.9% and from 1.8 to 32.0%, respectively, with significant differences among frozen batches. Rates of cleavage and blastocyst formation were significantly related to the nonreturn rate (r = 0.59, P < 0.001; r = 0.35, P < 0.05, respectively). The interaction between cleavage and blastocyst rate was 0.69 (P < 0.001). Significant variations (P < 0.05) among frozen semen batches within 15 bulls with >/= 2 different semen batches were found for the nonreturn rate (13.3%) of 2 bulls, for cleavage rates (26.7%) in 4 bulls and for blastocyst rates (20.0%) in 3 bulls. Significant differences (P < 0.05) among replicates within the 40 frozen semen batches were only found in 3 batches (7.5%) for the cleavage rate and in 7 batches (17.5%) for blastocyst rate. Overall, bull and frozen semen batch were the greatest sources of variation in the cleavage rate (30.6 and 29.4%, respectively), while testing date was the greatest source of variation in the blastocyst development rate (21.7%). The results indicated that in vitro fertilization and, to a lesser extent, culture to the blastocyst stage could be useful in estimating the potential fertilizing ability of frozen-thawed semen from dairy bulls.