Fertility effects of the 14;20 Robertsonian translocation in cattle

Superovulation and embryo collection procedures were used to study the effect of the 14;20 Robertsonian translocation on fertility and embryo viability. Karyotypes were successfully completed on cells from 77 of the 279 embryos prepared for such analysis. Embryos from 4 cows heterozygous for the translocation were studied. Two bulls with the same condition were studied by using their semen in artificial insemination of cows with normal karyotypes. The proportions of fertilized ova and transferable embryos were not different between cows with the 14;20 translocation and those with normal karyotypes, indicating that fertilization rates were not affected by the translocation. Twenty-two percent of the embryos which were karyotyped had an unbalanced karyotype and would theoretically not have survived to term. All of the theoretically predicted chromosome complements from such a translocation were observed as were three 58,XX,t karyotypes and a 58,XX karyotype. There was no difference in the percentage of embryos with abnormal karyotypes whether the cow or bull was the carrier. Results therefore indicate that fertility is rather severely impaired in carriers of the 14;20 translocation, as was observed with the 1;29 translocation, with most loss due to embryo mortality rather than a lowered conception rate.     

Additional evidence of the formation of unbalanced embryos in cattle with the 7/21 Robertsonian translocation

To confirm the effect of the 7 21 Robertsonian translocation on fertility in Japanese Black Cattle, cytogenetic studies were performed on embryos collected from the following 3 mating groups: normal bull cross normal cow, translocation carrier bull cross normal cow, and normal bull cross translocation carrier cow. All the analyzable embryos showed normal chromosome complements when the parents had a normal karyotype. In the group sired by the 7 21 translocation heterozygous bulls, a total of 56 embryos had metaphases suitable for chromosome analyses. Out of these embryos, 28 had normal chromosome complements and 25 were embryos with a balanced karyotype. However, 3 (5.4%) were monosomic and trisomic embryos, presumably resulting from the fertilization of normal ova by aneuploid spermatozoa. Unbalanced embryos were also observed in the chromosome analyses of embryos derived from the 7 21 translocation heterozygous cows. These results suggest that the 7 21 translocation in the heterozygous state may be associated with a slight reduction in reproductive efficiency.     

Use of semen from a bull heterozygous for the 1 29 translocation in an IVF program

In cattle, a translocation of the Robertsonian type between the largest and smallest chromosome leads to a reduction in fertility. This is substantiated by reduced nonreturn rates in daughter groups of bulls carrying the 1 29 translocation and in the heterozygous bulls themselves. This reduction in fertility is thought to be due to the early death of embryos with unbalanced karyotypes. The influence of semen from a bull known to be heterozygous for the 1 29 translocation on the outcome of a bovine IVF program was investigated. There was a significant difference (P<0.005) in terms of cleavage rate (59.8 vs 71.1%) and blastocyst rate (12.0 vs 20.0%) between the carrier and control bull, respectively. There was no difference in blastocyst quality as measured by cell number. The results observed in vitro are consistent with the field fertility records of the 2 bulls in terms of nonreturn rates (59.2 vs 70.6%, for the carrier and control bull, respectively).     

Status of oocytes and embryos of X-autosome translocation carrier sows

The impact of an X-autosome translocation t(Xp+; 14q-), on ovulation, fertilization and embryo survival in carrier sows, was examined and compared with these parameters of normal sows. Corpora lutea counts during week-2 and week-4 of gestation were similar in normal and carrier sows (14.4 +/- 1.36 and 15.5 +/- 2.18) although embryo recovery (11.0 +/- 1.87 and 6.0 +/- 1.47) was lower than that from normal sows (12.8 +/- 1.46 and 11.5 +/- 0.87), at these stages. Among the embryos karyotyped from the week-2 embryos of carrier sows, 42% were normal, 26.4% were carriers and 31.6% were of unbalanced chromosome make-up, and of the week-4 embryos of carriers, 33.3% were normal, 57.1% were carriers and 9.1% were chromosomally unbalanced females. The preponderance of females among the unbalanced embryos recovered at week-2 of gestation (11_ and 1_) and the total absence of males among those recovered at week-4, suggest that oocytes with unbalanced chromosome constitution are eliminated before week-2 of gestation if they are fertilized by Y bearing sperm, and that the unbalanced oocytes fertilized by X bearing sperm survive up to the peri-attachment stage even though all chromosomally unbalanced embryos are eliminated before term regardless of their sex.     

Monosomy and trisomy in bovine embryos sired by bulls heterozygous for the 1 29 Robertsonian translocation chromosome

Mature cows (n = 37) were superovulated and inseminated with semen from either bulls heterozygous for the 1 29 Robertsonian translocation or bulls with a normal karyotype. A total of 163 embryos was recovered, and preparations suitable for cytogenetic analysis were obtained from 67 (41.1%) of these. The karyotype was unbalanced in three (4.5%) of the embryos sired by semen from a bull carrying the translocation, whereas no karyotypic imbalance was recognized in the control group of embryos. Two of the unbalanced embryos were monosomic, while the third embryo was trisomic.     

A new centric fusion translocation in cattle: rob (13;19)

A new Robertsonian translocation has been found in cattle. A bull from Marchigiana breed (central Italy) was found to be a heterozygous carrier of a centric fusion translocation involving cattle chromosomes 13 and 19 according to RBA-banding and cattle standard nomenclatures. CBC-banding revealed the dicentric nature of this new translocation, underlining the recent origin of this fusion. In fact, both the bull's parents and relatives had normal karyotypes. In vitro fertilization tests were also performed in the bull carrying the new translocation, in two bulls with normal karyotypes (control) and in four other bulls carrying four different translocations.     

Cytogenetic studies of 1-29 translocation carrying cows and their embryos

An examination has been made of embryos from infertile 1-29 translocation carrying heifers. Cytogenetic measurements of C-banded chromosomes from these cattle, together with those from fertile 1-29 cows, have been made and compared with those from normal karyotypes. There are relative differences between normal animals and translocation carriers in the ratios of total to C-banded material throughout the karyotypes: their possible effects on mitosis and meiosis are discussed. Of the embryos recovered from the infertile heifers, one did not survive transplantation and others had various indications of inviability.     

Comparison of ovulation, fertilization and embryonic survival in low-fertility beef cows compared to fertile females

The objective was to determine physiological causes of low fertility in beef cows. Fertility was compared between low-fertility cows (34 British cows and 64 Brahman crossbred cows; cows that did not get pregnant when mated to fertile bulls in one or two previous breeding seasons); fertile cows (16 Brahman crossbreds; cows having a calf in several of the preceding breeding seasons), and virgin heifers (45 Brahman crossbreds, 2 yr of age). Females were mated to fertile bulls and killed 3 or 34 d after breeding to obtain reproductive tracts. There were no significant differences among groups in rates of ovulation or fertilization. Overall, 14% of females failed to ovulate and 24% that ovulated failed to undergo fertilization. The proportion of cows that were not detected in estrus before Day 34 of pregnancy was lower (P < 0.01) for low-fertility British cows (5 of 16 cows, 31%) than for other groups, including low-fertility Brahman crossbred cows (23/32, 72%), fertile cows (8/9, 89%), and heifers (21/24, 88%). All cows that did not return to estrus by Day 34 had an identifiable conceptus. The proportion of conceptuses recovered at Day 34 that were classified as normal (weight and length) was lower (P < 0.05) for cows with low fertility (British: 2/5, 40%; Brahman crossbred: 9/23, 39%) than for fertile cows (8/8, 100%) or heifers (18/21; 86%). Similarly, the proportion of cows in which a normal embryo was recovered (cows with normal embryos/number of cows mated) was lower (P < 0.001) for low-fertility British cows (2/16, 13%) and low-fertility Brahman crossbred cows (9/32, 28%) than for fertile cows (8/9, 89%) and heifers (18/24, 75%). In conclusion, cows that were infertile in previous breeding seasons did not experience reduced ovulation or fertilization rates, but had greater embryonic mortality. These data highlighted the importance of ovulation and fertilization failure and embryonic mortality as important determinants of pregnancy success. Moreover, increased embryonic loss after Day 34 contributed to infertility in low-fertility cows.     

Meiotic segregation analysis in cows carrying the t(1;29) Robertsonian translocation

Heterozygous carriers of Robertsonian translocations generally have a normal phenotype but present reproductive failure. In cattle, the t(1;29) Robertsonian translocation is very common and carriers show a 3-5% decrease in fertility. Some data suggest that female carriers have a higher decrease than male carriers but no direct studies of the chromosome content of oocytes from a t(1;29) carrier cow have been performed so far. Four heterozygous carrier cows underwent hormonal stimulations and follicles punctions and about 800 oocytes were matured in vitro. Six hundred metaphase II preparations were obtained and analysed by fluorescent in situ hybridization with bovine chromosome 1 and 29 painting probes. Proportions of different kinds of oocytes were assessed: 74.11% (292/394) were normal and balanced, 4.06% (16/394) unbalanced and 21.83% (86/394) diploid. For all cows, the number of normal oocytes was not significantly different from the number of translocated oocytes but the diploidy and unbalanced rate were significantly different between them. As found in bulls, the meiotic segregation pattern in cows has shown a preponderance of alternate products. However, the frequency of unbalanced gametes determined in females (4.06%) was significantly higher than the frequency observed in males (2.76%). The divergence in the rate of diploid gametes (0.04% vs. 21.83%) is mainly explained by the difference between males and females.     

Decreased fertility in related females heterozygous for the 1/29 chromosome translocation

Eighteen heifers and 120 cows which were descendants of a presumed 1/29 carrier Simmental bull were karotyped. Nine heifers (50%) and 48 cows (40%) were found to be heterozygous for the 1/29 translocation (59, XX, t(1q;29q)). The other animals were chromosomally normal (i.e., 60, XX) or not karotyped. The 48 1/29 cows were compared with 72 chromosomally normal cows with regards to days to first conception, calving interval, percentage of calves conceived, percentage of calves weaned and production efficiency (% calved conceived × % calved weaned). Nine carrier heifers were compared to the nine noncarrier heifers as to pregnancy status. Carrier, noncarrier and nonkarotyped relatives were compared to each other and to contemporary females with regard to pregnancy status at their initial exposure to males. The percentage of calves conceived (calving efficiency) in the 72 noncarrier and the 48 females heterozygous for the 1/29 translocation were 81.5 and 74.8%, respectively (P<0.07). Although days to first conception was longer and percentage of calves weaned and production efficiency were lower in the female heterozygous for the 1/29 translocation, the differences were not statistically different (P>0.10) from the noncarriers. Pregnancy rate was 44.4 and 66.7% (P>0.10) for nine carrier and nine noncarrier heifers, respectively. The pregnancy rate of carrier (65.4%), noncarrier (73.2%) and nonkarotyped (77.8%) relatives of this sire at their mating as yearlings, did not differ (P>0.10). The pregnancy rate as yearlings of carrier females (65.4%) and contemporary heifers (79.8%) did differ (P<0.05). Comparing the pregnancy rate as yearlings of all descendants (72.0%) of the Simmental sire to contemporary heifers (79.8%), a significant decrease (P<0.05) was found indicating that fertility of this sire may have been lower than other sires or that other factors beside the translocation affected fertility.     

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.     

Embryonic death in pigs caused by unbalanced karyotype

A landrace boar being heterozygous for a translocation has been used in 43 matings to 30 sows. Compared to a normal boar which also served part of the same sow group the defect boar had a by 30 % reduced fertility. Five different types of unbalanced karyotypes were detected in fetuses sired by the translocation heterozygote. Since no unbalanced karyotype was to be found postnatally in 111 offspring, all defect karyotypes seem to be lethal at the embryonic stage. Forty-one % of the postnatally investigated cases had the same translocation as the father.     

Embryonic development in repeat breeder and virgin heifers seven days after insemination

The aim of this study was to compare the development of embryos from repeat breeder heifers with that of embryos from virgin heifers at 7 days after standing heat. A total of 23 repeat breeder heifers (RBH) and 18 virgin heifers (VH) were utilized. The heifers were between 16 and 30 months of age and most of them were of the Swedish Red and White Breed. Two RBH were heterozygous for the $\text{1}\text{29}$ chromosome translocation, one RBH was a trisomy X and all the other heifers had normal karyotypes. All heifers were inseminated with frozen semen from the same bull and all inseminations were performed by the author. The fertility of the bull was above the average for the AI association to which it belonged. Embryos were collected by a non-surgical technique (89) or after slaughter (19). The morphology of the embryos was examined under a phase-contrast microscope and they were classified as being normal (N), morphologically deviating (MD) or degenerated (D). Thirteen embryos from RBH and 15 from VH were examined for total cell numbers after examination of their morphology.There was no significant difference in recovery rates of embryos between RBH (68%) and VH (76%) but independent of collection method the recovery rate of embryos from VH was numerically higher. The fertilization rate was high in both RBH (89%) and VH (97%). Seventyfour percent of the embryos collected from VH were normal ($\text{23}\text{31}$) while only 28% ($\text{11}\text{40}$) of the embryos collected from RBH had a normal morphology. The difference in number of normal embryos recovered from the two groups of heifers was highly significant (P < 0.005). Exclusion of the RBH heifers with deviating karyotype did not influence this difference. However, there was a tendency to a higher incidence of fertilization failure and morphologically deviating embryos in these heifers. The N embryos had significantly higher total cell numbers (P < 0.005) than the MD embryos but there was no significant difference in total cell numbers between N embryos from RBH or VH.The results of this study strongly indicate a higher incidence of abnormal embryos in RBH than in VH. It is likely that these deviations are followed by an increased incidence of early embryonic death.     

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: detection of translocations in embryos by fluorescence in situ hybridization

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.     

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but ininjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.     

Sperm nuclei analysis of 1/29 Robertsonian translocation carrier bulls using fluorescence in situ hybridization

In 1964, Gustavsson and Rockborn first described the 1/29 Robertsonian translocation in cattle. Since then, several studies have demonstrated the negative effect of this particular chromosomal rearrangement on the fertility of carrier animals. During the last decade, meiotic segregation patterns have been studied on human males carrying balanced translocations using FISH on decondensed sperm nuclei. In this work, we have applied the 'Sperm-FISH' technique to determine the chromosomal content of spermatozoa from two bulls heterozygous for the 1/29 translocation and one normal bull (control). 5425 and 2702 sperm nuclei were scored, respectively, for the two heterozygous bulls, using whole chromosome painting probes of chromosomes 1 and 29. Very similar proportions of normal (or balanced) spermatozoa resulting from alternate segregation were observed (97.42% and 96.78%). For both heterozygous bulls, the proportions of nullisomic and disomic spermatozoa did not follow the theoretical 1:1 ratio. Indeed, proportions of nullisomic spermatozoa were higher than those of disomic sperma tozoa (1.40% vs 0.09% (bull 1) and 1.29% vs 0.15% (bull 2) for BTA1, and 0.65% vs 0.40% (bull 1) and 1.11% vs 0.63% (bull 2) for BTA29). The average frequencies of disomic and diploid spermatozoa in the normal bull were 0.11% and 0.05%, respectively.     

Differences between Brahman and Holstein cows in response to estrus synchronization,superovulation and resistance of embryos to heat shock

Embryos from Bos indicus are more resistant to elevated culture temperature (i.e. heat shock) than embryos from some Bos taurus breeds. The present experiment was designed to determine if Brahman embryos have greater resistance to heat shock than Holstein embryos at a stage in development before the embryonic genome was fully activated. A second objective was to test breed effects on estrus synchronization and superovulation responses. A total of 29 Brahman and 24 Holstein cows were subjected to estrus synchronization using gonadotropin releasing hormone (GnRH) and prostaglandin F2alpha (PGF2alpha) superovulation. Embryos were collected at 48 h and day 5 after insemination. There was a tendency for a lower proportion of Brahmans to be detected in standing estrus than Holsteins. There were no differences between breeds in the proportion of cows detected in estrus using both tailpaint and standing estrus as criteria or in interval from PGF2alpha to estrus. The degree of synchrony in estrus was greater for Brahmans. Superovulation response was generally similar between breeds. At 48 h after insemination, there was a tendency for a greater proportion of Brahman oocytes to have undergone cleavage. Uncleaved oocytes were cultured for an additional 24 h-at this time, cleavage rate was similar between breeds. When embryos reached the 2-4-cell stage, they were heat-shocked for 4.5 h at 41 degrees C. This heat shock reduced the proportion of embryos that developed to the blastocyst stage but there was no breedxtreatment interaction. At day 5 after insemination, the number of embryos recovered was too low to allow comparison of breed effects. In conclusion, genetic effects on cellular thermotolerance that make Brahman embryos more resistant to heat shock are not expressed at the 2-4-cell stage. There were few differences between Brahman and Holstein in response to estrus synchronization and superovulation. The fact that cleavage tended to occur earlier in Brahman than Holstein embryos suggests breed differences in timing of ovulation, fertilization or events leading to cleavage.     

Selective elimination of chromosomally unbalanced zygotes at the two-cell stage in the Chinese hamster

The gametic and zygotic selection of genome imbalance was investigated in the Chinese hamster by direct chromosome analyses of spermatocytes and preimplantation embryos from crosses between chromosomally normal females and males heterozygous for a reciprocal translocation, T(2;10)3Idr, abbreviated here as T3. The karyotypes and the frequencies of embryos observed at the first cleavage in the cross +/+female X T3/+male were consistent with those expected from MII scoring in male T3 heterozygotes. Therefore, it was concluded that there was neither gametic selection against genome imbalance nor zygotic selection from fertilization until the first cleavage metaphase. However, 9.1-10.8% of embryos were arrested at the two-cell stage, and karyotypes of these embryos were confirmed as 22(2,10,10,10(2)), 21(2,10,10), and 21(2,10,10(2)). The common abnormality of these embryos was partial monosomy of chromosome 2. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of other segments of chromosomes 2 and 10, had fewer blastomeres than chromosomally balanced embryos. This finding suggests that cleavage of these embryos had been retarded by day 4 of gestation.     

The establishment and application of preimplantation genetic haplotyping in embryo diagnosis for reciprocal and Robertsonian translocation carriers

Background

Preimplantation genetic diagnosis (PGD) is now widely used to select embryos free of chromosomal copy number variations (CNV) from chromosome balanced translocation carriers. However, it remains a difficulty to distinguish in embryos between balanced and structurally normal chromosomes efficiently.

Methods

For this purpose, genome wide preimplantation genetic haplotyping (PGH) analysis was utilized based on single nucleotide polymorphism (SNP) microarray. SNPs that are heterozygous in the carrier and, homozygous in the carrier’s partner and carrier’s family member are defined as informative SNPs. The haplotypes including the breakpoint regions, the whole chromosomes involved in the translocation and the corresponding homologous chromosomes are established with these informative SNPs in the couple, reference and embryos. In order to perform this analysis, a reference either a translocation carrier’s family member or one unbalanced embryo is required. The positions of translocation breakpoints are identified by molecular karyotypes of unbalanced embryos. The recombination of breakpoint regions in embryos could be identified.

Results

Eleven translocation families were enrolled. 68 blastocysts were analyzed, in which 42 were unbalanced or aneuploid and the other 26 were balanced or normal chromosomes. Thirteen embryos were transferred back to patients. Prenatal cytogenetic analysis of amniotic fluid cells was performed. The results predicted by PGH and karyotypes were totally consistent.

Conclusions

With the successful clinical application, we demonstrate that PGH was a simple, efficient, and popularized method to distinguish between balanced and structurally normal chromosome embryos.

     

A comparison of natural service fertility of yearling and two-year-old bulls on pasture

The natural service fertility of yearling bulls on pasture was compared with that of 2-year-old bulls in a study (2 experiments) which involved a total of 748 composite breed cows over 3 breeding seasons. Other factors such as age of the cows, breed, year effects, and the sequence in which bulls of a particular age were assigned to cows were also evaluated. Pregnancy and calving rates and calving date were used as criteria for assessing fertility. The age of the bull, breed, year, or bull sequence did not significantly affect any of the traits studied. The age of cow was important in all the traits studied, with mature cows (5 years and older at breeding) calving earlier (P < 0.05) than the youngest group (2 years old at breeding) of cows, in both experiments. The pregnancy rate was also lower in the younger cows than in the mature cows in the first experiment. The results suggest that the age range of the cow herd is more important in determining herd fertility than the age of the bull or any of the parameters studied.