The costs of breed reconstruction from cryopreserved material in mammalian livestock species

The aim of this work was to compare costs, in the horse, cattle, sheep, swine, and rabbit species, for the creation of gene banks for reconstruction of an extinct breed, using different strategies: embryos-only, embryos in combination with semen, and semen-only. Three cost measures were used: time required for population reconstruction, cost for creation of the gene bank, number of years-keeping-female to reach reconstruction. Semen costs were estimated across four scenarios: the presence or absence of a commercial market for semen, purchase of semen donors, and semen extracted from the epididymus. The number of cells were doubled to take into account the creation of two storage sites. The strategy embryos-only required the shortest time to reach reconstruction. With the strategy embryos + semen, time increased with decreasing proportions of embryos. With semen-only, reconstruction time varied from 2 to 21 years. A high variation of costs was observed across species and strategies, from 360 Euros in the rabbit to 1 092 300 in the horse. In all species, the embryos-only strategy was about 10% more expensive than using 90% embryos + semen. Decreasing the percentage of embryos further diminished costs. The number of years-keeping-female ranged across strategies, from 2 in the rabbit, to a maximum of 12 878 in the horse.     

Semen parameters and level of microsatellite heterozygosity in Noriker draught horse stallions

It was the aim of the present study to determine physiological values for different semen parameters in an endangered draught horse breed, the Austrian Noriker. Because small population size is often believed to cause a decrease in fertility and/or semen quality through inbreeding and a reduction in genetic variation, the general genomic heterogeneity of the breed was estimated on the basis of microsatellite variation and correlated to semen parameters. Semen could be collected from 104 of 139 stallions with semen collection being more often successful in younger stallions. Mean volume of ejaculates was 90.8+/-55.1 ml, density 243+/-114 x 10(6)ml(-1), total sperm count 21.0+/-23.7 x 10(9), percentage of morphologically normal spermatozoa 38+/-18% and total motility 50+/-23%. Total sperm count and semen motility were significantly affected by age. Blood samples of 134 stallions were analysed for 12 microsatellite DNA markers. Genotypes of 110 stallions with at least 11 successfully typed markers were used for calculation of heterozygosity. A total of 82 alleles was identified with a mean of 6.8 alleles per marker. Heterozygosity varied between 35 and 76% for the different markers, mean heterozygosity was calculated to 63%. No correlation between heterozygosity and semen parameters was found.     

Intracytoplasmic sperm injection (ICSI) in small ruminants

In this study, a linear programming model was developed to maximize the gross margin of milk production by determining the optimal use of different reproductive technologies in a dairy herd. The model has the potential to vary the use of conventional artificial insemination, insemination with X-sorted sperm, and the use of unselected or sex-selected embryo recovery and transfer. Data from Finnish dairy herd recording systems were used to parameterize the model. This paper presents the results of 6 scenarios for a herd size of 60 dairy cows. In the basic scenario, the optimum economic combination for Finnish conditions was to inseminate 10 heifers and 22 cows with unsorted semen, 8 heifers with X-sorted sperm, and to use 20 cows as embryo donors which was the upper constraint for this technique. The embryo donors were inseminated with conventional semen for both embryo production and their subsequent pregnancy. Without restriction on embryo recovery, the optimum combination was to use all heifers as donors of sex-selected embryos and all cows as donors of unselected embryos. It was more profitable to produce female embryos with X-sorted sperm than by sorting embryos. Embryo recipients were not economically justified in any scenario. In practice, the optimal strategy is herd-specific depending on the input costs, output values and the technical success of each reproductive technology in that herd. This single-year linear programming model adequately differentiates between breeding technologies within a herd, but further research is needed to develop dynamic models to consider genetic improvement and herd expansion.     

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

A protocol for the cryoconservation of breeds by low-cost emergency cell banks - a pilot study

A protocol was developed for collection and storage of somatic cell samples under adverse conditions with little infrastructure, for somatic-cell banks as a backup for endangered livestock breeds. The procedure, which is uniform across species, includes sample collection with ear taggers with an integrated tag/vial system, and recording of global positioning system (GPS) coordinates, digital images and breed information. In a pilot study, the procedures were tested on six local Vietnamese populations of pigs, sheep and goats. Initial investment was around €3000, while the total variable cost for sampling one breed with 25 females and 25 males was less than €1000. With support from local organisations, the sampling of six breeds with 300 animals was carried out in 2 months. The protocol and the complete workflow for setting up a somatic-cell bank, together with data collection, are described. The procedure has proved practicable and exceedingly cheap relative to the cryopreservation of semen or embryos.     

Effects of different concentration and combinations of cryoprotectants on sperm quality,functional integrity in three Indian horse breeds

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4–6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.     

Fertility of fresh and frozen-thawed goat semen during the nonbreeding season

A total of 4109 does of a local Greek breed (Capra prisca) were synchronized with intravaginal MPA-sponges and PMSG, and 24 bucks of Alpine (n = 8), Saanen (n = 8) and Damascus (n = 8) breeds were used for studying the fertility of nonfrozen and frozen-thawed semen during the nonbreeding season (June to August). Artificial insemination (AI) was performed once (50 to 55 h after sponge withdrawal) or twice (36 and 60 h after sponge withdrawals with fresh semen (collected during the nonbreeding season, stored at 16 degrees C and inseminated within 6 h) or frozen semen (prepared from the same bucks during the preceding breeding season). The induction of estrus was successful, varying between 91.0 and 95.0%. The form of semen (fresh or frozen-thawed used for inseminating the synchronized does affected their fertility: the overall kidding rate with fresh semen (65.5%) was higher (P < 0.05) than that with frozen-thawed semen (53.4%). The fertility level was also affected by the number of inseminations performed: the overall kidding rate was significantly higher (P < 0.001) in the does inseminated twice with fresh or frozen-thawed semen (70.4 and 59. 1%, respectively) than in those inseminated only once (48.9 and 44.9%, respectively). Finally, the breed of the buck used for preparing the fresh or the frozen-thawed semen affected the fertility level of the does. The kidding rate was higher in does inseminated with fresh semen prepared from bucks of the Damascus breed than from bucks of Saanen or Alpine breed. However, when frozen-thawed semen was used the kidding rate was lower in does inseminated with semen prepared from bucks of the Damascus breed than from bucks of the Alpine or Saanen breed. It is concluded that the fresh semen of Alpine, Saanen and Damascus breed bucks, born and raised under the climate conditions prevailing in Greece (34 degrees to 41 degrees N), can be used successfully during the nonbreeding season (June to August) for inseminating does.     

Genetic and non-genetic parameters of several characteristics of production and semen quality in young bucks

The objective of this study was to evaluate genetic and non-genetic factors influencing characteristics of young buck semen production using a multivariate model that takes into account the longitudinal structure of data. Data were collected from 1989 to 2002 at two French A.I. centres. The data corresponded to 13151 and 9206 ejaculates of 758 Alpine and 535 Saanen bucks respectively, collected at the beginning of the first breeding season (September-December). The semen volume, the total number of spermatozoa, the concentration, the motility score of spermatozoa after freezing and the percentage of motile spermatozoa after freezing were registered for each ejaculate. Within-breed heritabilities and repeatabilities were estimated using a multivariate animal model using a power spatial covariance structure for environmental effect. For all characteristics and the two breeds, the main source of variation was the year-month interaction that interacted with the centre. We observed a decrease in years of motility score after freezing. Age and frequency of collection had a significant effect on semen volume and number of spermatozoa for both breeds, and on concentration of spermatozoa for the Alpine breed. No effect of these factors was found on the characteristics observed after freezing. Heritabilities for concentration, number of spermatozoa, semen volume, motility score after freezing and percentage of motile spermatozoa after freezing per ejaculate were respectively, 0.32, 0.15, 0.25, 0.12 and 0.05 for the Saanen breed and 0.34, 0.25, 0.29, 0.17 and 0.03 for the Alpine breed. Genetic correlations between volume and number of spermatozoa were respectively, 0.74 for the Alpine breed and 0.86 for the Saanen breed. Further study is required to compare the semen characteristics of young bucks with their mature production.     

Impact of the use of cryobank samples in a selected cattle breed: a simulation study

Background

High selection pressure on domestic cattle has led to an undesirable increase in inbreeding, as well as to the deterioration of some functional traits which are indirectly selected. Semen stored in a cryobank may be a useful way to redirect selection or limit the loss of genetic diversity in a selected breed. The purpose of this study was to analyse the efficiency of current cryobank sampling methods, by investigating the benefits of using cryopreserved semen in a selection scheme several generations after the semen was collected.

Methods

The theoretical impact of using cryopreserved semen in a selection scheme of a dairy cattle breed was investigated by simulating various scenarios involving two negatively correlated traits and a change in genetic variability of the breed.

Results

Our results indicate that using cryopreserved semen to redirect selection will have an impact on negatively selected traits only if it is combined with major changes in selection objectives or practices. If the purpose is to increase genetic diversity in the breed, it can be a viable option.

Conclusions

Using cryopreserved semen to redirect selection or to improve genetic diversity should be carried out with caution, by considering the pros and cons of prospective changes in genetic diversity and the value of the selected traits. However, the use of genomic information should lead to more interesting perspectives to choose which animals to store in a cryobank and to increase the value of cryobank collections for selected breeds.     

Reconstruction of genetic distance matrix

A method for reconstruction of genetic distances' matrix, based on linear combination of physical distances' matrices among populations and mean sizes of the population matrices is proposed. The analogue of genetic distances' matrix obtained correlates with the matrix at the level 0.59. The reconstruction may be used for the populations of about 2-3 neighbour districts. An index xi is introduced, as a constant describing some big regions. Comparison of reconstructed matrix of genetic distances with some well-known matrices of genetic distances is performed.     

Influence of sire and sire breed (Gyr versus Holstein) on establishment of pregnancy and embryonic loss in lactating Holstein cows during summer heat stress

Heat stress has negative effects on pregnancy rates of lactating dairy cattle. There are genetic differences in tolerance to heat stress; Bos taurus indicus (B. t. indicus) cattle and embryos are more thermotolerant than Bos taurus taurus (B. t. taurus). In the present study, the effects of sire and sire breed on conception and embryonic/fetal loss rates of lactating Holstein cows during the Brazilian summer were determined. In Experiment 1, cows (n=302) were AI after estrus detection or at a fixed-time with semen from one Gyr (B. t. indicus) or one Holstein sire (B. t. taurus). Pregnancy was diagnosed 80 days after AI. In Experiment 2, cows (n=811) were AI with semen from three Gyr and two Holstein sires. Pregnancy was diagnosed at 30-40 and at 60-80 days after AI. Cows diagnosed pregnant at the first examination but non-pregnant at the second were considered as having lost their embryo or fetus. Data were analyzed by logistic regression. The model considered the effect of sire within breed, sire breed, days postpartum, period of lactation, and AI type (AI after estrus versus fixed-time). There was no effect of the AI type, days postpartum or milk production on conception or embryonic loss rates. The use of Gyr bulls increased pregnancy rate when compared to Holstein bulls [9.1% (60/657) versus 5.0% (23/456), respectively, P=0.008; data from Experiments 1 and 2 combined]. Additionally, in Experiment 2, cows inseminated using semen from sire #4 (Gyr) had lower embryonic loss (10%) when compared with other B. t. indicus (35.3% and 40%) or B. t. taurus sires (18.2% and 38.5%, P=0.03). In conclusion, the use of B. t. indicus sires may result in higher conception rates in lactating Holstein cows during summer heat stress. Moreover, sire can affect embryonic loss and selection of bulls according to this criterion may result in higher parturition rates in lactating Holstein cows.     

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.     

Developmental capacity of ferret embryos by nuclear transfer using G0/G1-phase fetal fibroblasts

With the ultimate goal of establishing experimental protocols necessary for cloning ferrets, the present study has established parameters for the reconstruction of ferret embryos by nuclear transfer (NT) using G0/G1-phase donor fetal fibroblasts. Cumulus-oocyte complexes were harvested from superovulated ferrets and cultured in maturation medium for 24 h. Matured oocytes were then enucleated and injected with the fibroblast nuclei derived from 14-16-h serum-starved cells. Reconstructed embryos were then activated by a combination of electric pulses and chemical stimulations. Subsequently, the reconstructed and activated embryos were either cultured in vitro or transferred to pseudopregnant ferrets to evaluate their developmental capacity in vitro and in vivo. Our results demonstrated that 56.3% of reconstructed embryos (n = 187) cleaved, while 26.0% and 17.6% developed to morula and blastocyst phases in vitro, respectively. The blastocysts derived from NT embryos demonstrated normal morphology by differentially staining as compared to normal blastocysts developed in vivo following fertilization. In vivo developmental studies at 21 days posttransplantation demonstrated 8.8% of reconstructed embryos (n = 91) implanted into the uterine lining of recipients, while 3.3% formed fetuses. However, reconstructed embryos (n = 387) failed to develop to term (42 days). These results demonstrate donor nuclei of G0/G1-phase fetal fibroblast cells can be reprogrammed to support the development of reconstructed ferret embryos in vitro and in vivo; however, a significant third-trimester block occurs preventing full-term development.     

Activation of equine nuclear transfer oocytes: methods and timing of treatment in relation to nuclear remodeling

Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h after reconstruction. Four activation treatments were evaluated at each time point: injection of sperm extract alone, or in combination with 6-dimethylaminopurine (6-DMAP), cytochalasin B, or 1% dimethylsulphoxide. There was no significant difference in normal cleavage rate or average nucleus number of embryos between equine oocytes activated <30 min or at 2 h after reconstruction. The combination of 6-DMAP with sperm extract significantly (P < 0.01) improved cleavage rate compared with the other three treatments. Activation with sperm extract and 6-DMAP 2 h after donor nucleus injection gave the highest cleavage (79%) and the highest cleavage with normal nuclei (40%). Sperm extract and 6-DMAP also effectively activated oocytes parthenogenetically, yielding 83% cleavage and 73% cleavage with normal nuclei. These results indicate that although nuclear remodeling occurs rapidly in equine cytoplasts, early activation does not improve embryonic development after reconstruction.     

Retrospective study of factors affecting multiple ovulations,embryo recovery,quality, and diameter in a commercial equine embryo transfer program

In this study, 198 donor mares of different breeds, ages, and reproductive category were inseminated with fresh, cooled and frozen or frozen and cooled semen at the embryo transfer station or in private artificial insemination centers during 10 breeding seasons. The results of this activity were retrospectively analyzed by Pearson Chi-square test and logistic regression to evaluate factors affecting multiple ovulations, embryo recovery, embryo quality, and embryo diameter. Out of the 661 cycles, 937 ovulations were recorded (mean ovulations/cycle: 1.42 ± 0.58). Ovulation rate and incidence of multiple ovulations were significantly affected by age, breed, and reproductive category. Uterine flushings for embryo recovery were performed between 7 and 10 days after ovulation and resulted in the recovery of 338 embryos (51.1% embryos/cycle and 36.1% embryos/ovulation, respectively). At least one embryo was recovered in 298 flushings (45.1%). The factors affecting embryo recovery were age, breed, reproductive category, type of semen, number of ovulations, and location of artificial insemination. Flushing protocol and day of flushing had no effect on embryo recovery. Age, type of semen, number of ovulations, and day of flushing had a significant influence on embryo diameter (N = 215). None of the factors included in the model had an effect on embryo quality distribution.     

Embryo cloning in sheep: work in progress

We summarize here the procedures for nuclear transfer using S-phase cytoplasts and describe a new method for avoiding loss of reconstructed embryos from the oviducts during in vivo culture. We obtained 2 clones of 5 genetically identical animals following the transfer of blastomeres from 16-cell embryos into enucleated preactivated cytoplasts. Metaphase II oocytes and embryos were surgically collected from superovulated Sarda breed ewes 54 and 120 h after sponge removal, respectively. Oocytes were exposed for 15 min to 5 mug/ml of Hoechst 33342 and were micromanipulated at room temperature. Efficiency in embryo reconstruction was 100% for enucleation and 98% for fusion. Embryos were embedded in agar as separate clones and transferred into the oviducts of temporary recipients. The fimbriae were closed with glass-nylon made filters. Embryo recovery from the temporary recipients was 97.3%, with a cleavage rate of 81.4%; development to morula-blastocyst stage was 70.6%. A total of 29 Grade 1 blastocysts corresponding to 5 clones were transferred into 13 naturally synchronous ewes, and scanning was performed at 30 and 90 d. Ten ewes were pregnant at the first scanning and nine at the second for a final pregnancy rate of 71.4%; the survival rate at term was 48%. Overall, we obtained 4 clones of identical lambs: two sets of 5 (one male set and one female set) and two sets of twins (both sets male). Pregnancy length in recipients carrying clones was longer than the standard period in Sarda breed (153 vs 150 d, respectively). Weight at birth was higher for male lambs obtained from nuclear transfer than for normal males (4.1 vs 3.6 kg), while the weight for females was normal.     

Effect of cytoplast on the development of inter-subspecies nuclear transfer reconstructed goat embryo

The aim of this study was to investigate effect of cytoplast on the development competence of reconstructed embryos derived from inter-subspecies somatic cell nucleus transfer (SCNT). First, the development potency of reconstructed embryos produced by transferring Boer goat fibroblast cell nucleus of different ages into enucleated Sannen goat ova was evaluated in order to determine which age of nuclear donor is favorable for the reconstructed embryos development. Secondly, the another component of reconstructed embryos, "cytoplast," was evaluated by comparing the effect of ovum cytoplast derived from Sannen male symbol x Boer female symbol descendant on the reconstructed embryos development to that of Sannen goat ovum cytoplast. The results revealed that the development rate of the reconstructed embryos derived from 2 months old Boer goat somatic cells was the highest, their gestation rate was up to 50%, and one viable male offspring was obtained. The cytoplast derived from the crossbreeding goats improves the development competence of reconstructed embryos, which birth rate was 5.5%. The genetic identification of offspring by using PCR-SSCP analysis confirmed that these cloned kids were derived from the donor. The results above reveal that the cytoplast of Sannen goat ovum could induce the dedifferentiation of somatic cell nuclei derived from Boer goat, but the reprogramming process of these reconstructed embryos seems incomplete, probably due to some incorrect processes happened after implantation. Relatedness components of nucleus donor in cytoplast of the crossbreeding goat may be helpful to induce the dedifferentiation of somatic cell nuclei completely and improve the development competence of the reconstructed embryos.     

Estimation of genetic parameters of semen characteristics and reproductive traits in AI boars

(Co)variance components and further genetic parameters of boar semen characteristics and reproductive traits were estimated using the REML procedure applied to multi-trait animal models. The calculations were based on data from 210,733 ejaculates stemming from 2862 AI boars and collected from 1990 to 1997 in insemination stations for boars in the Czech Republic. Equal model equations for all traits included the AI station and the breed or breed combination as fixed effects, the interval between two collections for the boar as covariable and the animal and residual effects as random effects. The following heritabilities were estimated: semen volume 0.58, sperm concentration 0.49, progressive motion of spermatozoa 0.38, abnormal spermatozoa 0.34, number of total spermatozoa 0.42, number of insemination doses 0.40, number of piglets born alive 0.08, total number of piglets born 0.05 and conception rate 0.29. Heritabilities and genetic correlations were estimated on average values for each boar.     

Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.