Sex ratios in dairy cattle under various conditions

Current calving information was obtained on 35,102 single births in 2254 dairy herds. The overall proportion of males to females was 50.8%. The 5 dairy breeds did not differ. Only 6 of 111 sires studied produced calves with a sex ratio different from breed average at P≤0.5. This is the number expected by chance alone. A slight bias seems to occur when reporting the sires of the cows according to the sex of the cow's calf. The sex ratio deviated from expected in a small sample of repeat breeder cows, but when a new and larger sample of 2,084 such cows which calved was obtained, there was no change associated with service number. The time of insemination was recorded for 12,764 heifers and cows first seen in estrus in the morning and 4,799 animals first seen in estrus in the evening. There was no effect of time of insemination on sex ratio. Likewise there was no effect of age of cows or season of breeding on sex ratio at birth. Because the sex ratio for cows requiring one insemination per pregnancy was not different from repeat breeders it is suggested that the sex ratio at fertilization and birth may be similar.     

Human sex ratio as a function of the timing of insemination within the menstrual cycle: a review

A review of the beliefs and research results on the influence of the time of insemination on the sex ratio is presented. The ancient Greek notion that more males were produced by early postmenstrual insemination was supported as late as the early 1900s, although, by then, that belief was not uncontested. The view soon changed to the one of Dechman in which midcycle insemination favored the birth of males because of the deterioration of the ovum and other dominance/submission arguments. There were also some 20th century writers who recommended premenstrual insemination for producing male offspring. More recent thinking, taking advantage of new knowledge in reproductive physiology, holds that insemination immediately prior to ovulation favors males, whereas earlier insemination favors females. Artifical insemination of women 3 or more days before ovulation has been reported to result in an excess of females, while natural insemination during the same time-frame produced an excess of males. More clinical research, utilizing a uniform methodology for determining the time of ovulation, is needed to elucidate the relationship between the date of insemination, menstrual-cycle day, and sex outcome to the sex ratio. Possible causes for natural variations in the sex ratio should also be investigated.     

Insemination factors related to timed AI in cattle

Six-day-old bovine ova/embryos were recovered non-surgically and used as biomonitors to evaluate time of artificial insemination. These embryos/ova provided information regarding fertilization status and embryo quality, as well as quantitative and qualitative data regarding associated accessory sperm. Both sperm access to the ovum (addressed by accessory sperm) and fertilization status/embryo quality were important in addressing pregnancy rate for specific intervals from the onset of estrus to insemination. Based on these biomonitors, early insemination failed to achieve optimum pregnancy rate due to inadequate access of sperm to the ovum (i.e., low fertilization rate, manifested by low accessory sperm numbers). However, embryo quality was high in early inseminations, which favors pregnancy. Late insemination failed to achieve optimum pregnancy rate (due to reduced embryo quality), however, sperm access to the ovum was highest. Thus, the selection of an insemination time to achieve optimum pregnancy rate appeared to be a compromise between the two extreme intervals. For timed-AI programs, consideration of the time of ovulation (and its variability) becomes important, in addition to conventional considerations, such as semen handling, site of insemination, and bull selection.     

Fertilization efficiency of in vitro matured oocytes transferred to oviducts of inseminated goats: a model to assess in vivo fertilization performance of goat spermatozoa

An alternative to conventional in vivo validation of sperm assays might be to assess the fertilization rate of multiple oocytes transferred to the oviducts of inseminated females. Increasing the number of oocytes increases the egg-sperm ratio in the oviduct under an unaltered endocrine milieu, setting the basis for picking up statistical differences between treatments in small populations. The study evaluated the model by transferring oocytes to females inseminated under conditions that are known to modify the fertilization rate in the field. The study then evaluated the use of cattle oocytes to replace goat oocytes for assessing sperm function under this model. In Experiment 1, 12 females were inseminated at estrus with either 100 or 300 million spermatozoa 20 h before transferring homologous oocytes into the oviduct ipsilateral to the ovulation point. In Experiment 2, 10 females were inseminated either once or twice; 10-20 h later, homologous oocytes were transferred into the oviduct ipsilateral to the ovulation point. In Experiment 3, 13 bilateral-ovulated females were inseminated and 20 h later goat and cattle oocytes were transferred to contralateral oviducts. Then, 16-20 h later, oocytes were flushed from the oviduct, cleaned of spermatozoa and stained to assess the fertilization rate. The fertilization rate was improved by increasing sperm numbers at insemination (P < 0.04) and by increasing the number of inseminations (P < 0.02). The results in Experiment 3 showed that fertilization rates were similar for goat and cattle oocyte (P > 0.05) and that fertilization values were highly correlated (r = 0.811, P < 0.001). Results suggest that the model can be used for in vivo validation of in vitro sperm assays by facilitating the expression of statistical differences in small number of animals. In addition, cattle oocytes can be used to replace goat oocytes to study in vivo sperm function in goats.     

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

Effect of the interval between estrus onset and artificial insemination on sex ratio and fertility in cattle: a field study

We have carried out a field trial in cattle to study the effect of the interval between the onset of estrus and AI on sex ratio and fertility. Data were obtained from 716 cows that had been inseminated at different times between 8 and 44 h from the visual detection of estrus. Before analyzing the data, it was grouped in three intervals considering the time between estrus onset and AI (8-18, 18-30, and > or = 30 h). Our results show that the percentage of calved females (73.05%) is significantly superior for early inseminations (8-18 h), and it decreases 1.85% per hour from the onset of estrus. Delayed AIs (> or = 30 h) produce a significant deviation of the sex ratio towards the males (72.06%); nevertheless, fertility (percentage of successful pregnancies) diminishes significantly, from 66.19% (8-18 h) to 45.35% (> or = 30 h). In conclusion, variations in the interval between the onset of estrus and AI modify sex ratio. However, we must consider its effect on fertility.     

Effect of timing of artificial insemination on gender ratio in beef cattle

It was recently reported that cows inseminated at approximately 10 or 20 h before an expected ovulation deliver predominately a bull or heifer calf, respectively. The objective of this study was to further investigate the effect of timing of insemination on the gender of offspring in cattle. Angus heifers (n = 41) and cows (n = 98) were used in the study. Heifers were synchronized with a 16-d treatment of melengestrol acetate followed 17 d later with an injection of PGF2alpha. Cows were synchronized with GnRH followed 7 d later with PGF2alpha. A HeatWatch electronic estrus detection system was used to determine the onset of estrus. Based on previous studies, it was assumed that ovulation occurs approximately 32 h after the onset of estrus. Therefore, animals were artificially inseminated at either 8 to 10 h (early; > or = 20 h before expected ovulation) or 20 to 25 h (late; < or = 10 h before expected ovulation) after the onset of estrus. Sixty to 80 d after insemination, ultrasonography was used to confirm pregnancy status and to determine the gender of fetuses. Gender of calves was subsequently confirmed at calving. Data were analyzed for effects of time of insemination and sire or semen batch on gender ratio, as well as any effect of length and/or intensity of estrus on conception rate and gender ratio. Twenty-nine of 41 heifers and 69 of 98 cows were detected in estrus after synchronization and were inseminated; 20 of 29 heifers and 48 of 69 cows were subsequently confirmed pregnant. Neither the length of estrus nor its intensity (number of mounts) had an effect on pregnancy rate or gender ratio (P > or = 0.418). Timing of insemination (early versus late) had no effect on gender ratio (P = 0.887). Semen from 13 sires representing 17 lots was used to inseminate the cows and heifers. No differences (P = 0.494) were detected in the gender ratios resulting from different sires or semen batches. In contrast to previous findings, our results indicate that inseminating beef cattle at approximately 20 or 10 h before an expected ovulation does not alter the gender ratio of the resultant calves.     

History of commercializing sexed semen for cattle

Although the basic principles controlling the sex of mammalian offspring have been known for a relatively long time, recent application of certain modern cellular methodologies has led to development of a flow cytometric system capable of differentiating and separating living X- and Y-chromosome-bearing sperm in amounts suitable for AI and therefore, commercialization of this sexing technology. After a very long history of unsuccessful attempts to differentiate between mammalian sperm that produce males from those that produce females, a breakthrough came in 1981 when it was demonstrated that precise DNA content could be measured. Although these initial measurements of DNA content killed the sperm in the process, they led to the ultimate development of a sperm sorting system that was capable, not only of differentiating between live X- and Y-sperm, but of sorting them into relatively pure X- and Y-sperm populations without obvious cellular damage. Initial efforts to predetermine the sex of mammalian offspring in 1989 required surgical insemination, but later enhancements provided sex-sorted sperm in quantities suitable for use with IVF. Subsequent advances in flow sorting provided minimal numbers of sperm sufficient for use in AI. It was not until the flow cytometric sorting system was improved greatly and successful cryopreservation of sex-sorted bull sperm was developed that efficacious approaches to commercialization of sexed semen could be implemented worldwide in cattle. A number of companies now offer sex-sorted bovine sperm. Innovative approaches by a diverse group of scientists along with advances in computer science, biophysics, cell biology, instrumentation, and applied reproductive physiology provided the basis for commercializing sexed semen in cattle.     

Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially?

It has been reported that the mammalian female could have a preconceptual influence on the sex of her offspring, and it has been hypothesized that this influence could go some way toward accounting for the reported lower fertility following insemination with sex-sorted sperm. To test whether in vitro matured oocytes are able to select X- or Y-bearing spermatozoa following in vitro fertilization (IVF), we fertilized in vitro 1788 oocytes with X-sorted semen, Y-sorted semen, a mix of X- and Y-sorted semen, and unsorted semen from the same bull, and cultured until Day 9. Fertility was assessed by recording cleavage rate at 48 h postinsemination (hpi) and blastocyst development until Day 9. Embryos were sexed at the two- to four-cell stage and the blastocyst stage. The proportion of zygotes cleaving at 48 hpi was not different between X- and Y-sorted groups and the mix of X- and Y-sorted semen group; however, all were significantly lower than the unsorted group (P < 0.001). Blastocyst yield on Day 6 was significantly higher (P < or = 0.01) in the control group compared with the rest of the groups. Cumulative blastocyst yields on Days 7, 8, and 9 were also significantly higher (P < or = 0.01) in the unsorted group compared with the sorted groups. The proportion of female and male two- to four-cell embryos obtained following IVF with X- and Y-sorted sperm was 88% and 89%, respectively and the sex ratio at the two- to four-cell stage was not different following IVF with unsorted or sorted/recombined sperm (56.9% males vs. 57% males, respectively). At the blastocyst stage, similar percentages were obtained. In conclusion, the differences in cleavage and blastocyst development using sorted versus unsorted sperm are not due to the oocyte preferentially selecting sperm of one sex over another, but are more likely due to spermatic damage caused by the sorting procedure.     

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.     

Female sperm storage mediates post‐copulatory costs and benefits of ejaculate anticipatory plasticity in the guppy

Males of many species evolved the capability of adjusting their ejaculate phenotype in response to social cues to match the expected mating conditions. When females store sperm for a prolonged time, the expected fitness return of plastic adjustments of ejaculate phenotype may depend on the interval between mating and fertilization. Although prolonged female sperm storage (FSS) increases the opportunity for sperm competition, as a consequence of the longer temporal overlap of ejaculates from several males, it may also create variable selective forces on ejaculate phenotype, for example by exposing trade‐offs between sperm velocity and sperm survival. We evaluated the relationship between the plasticity of ejaculate quality and FSS in the guppy, Poecilia reticulata, a polyandrous live‐bearing fish in which females store sperm for several months and where stored sperm contribute significantly to a male's lifelong reproductive success. In this species, males respond to the perception of future mating opportunities by increasing the quantity (number) and quality (swimming velocity) of ready‐to‐use sperm (an anticipatory response called ‘sperm priming’). Here we investigated (a) the effect of sperm priming on in vitro sperm viability at stripping and its temporal decline (as an estimate of sperm survival), and (b) the in vivo competitive fertilization success in relation to female sperm storage using artificial insemination. As expected, sperm‐primed males produced more numerous and faster sperm, but with a reduced in vitro sperm viability at stripping and after 4 hr, compared with their counterparts. Artificial insemination revealed that the small (nonsignificant) advantage of primed sperm when fertilization immediately follows insemination is reversed when eggs are fertilized by female‐stored sperm, weeks after insemination. By suggesting a plastic trade‐off between sperm velocity and viability, these results demonstrate that prolonged female sperm storage generates divergent selection pressures on ejaculate phenotype.     

哺乳动物精子分离方法的研究进展

分离X、Y精子,用于人工授精或体外受精,是目前实现哺乳动物性别控制的最有效手段。本文对哺乳动物精子分离及分离精子纯度评估方法的研究历史及现状作一回顾和总结。     

Effect of time of artificial insemination on embryo sex ratio in dairy cattle

The objective of the present study was to examine whether different intervals between insemination and ovulation have an influence on the sex of seven-day-old embryos in dairy cattle. Cows were inseminated once with semen of one of two bulls of proven fertility between 36 h before ovulation and 12 h after ovulation. Time of ovulation was assessed by ultrasound at 4-h intervals. In total, 64 embryos were determined to be male or female. Of these 64 embryos, 51.6% were female. The sex ratio in the various insemination-ovulation intervals (early: between 36 and 20 h before ovulation; intermediate: between 20 and 8 h before ovulation; late: between 8 h before and 12 h after ovulation) did not significantly differ from the expected 1:1 sex ratio (50, 50 and 55% females, respectively). Bull (Bull A and B) and Parity (primiparous and multiparous) had no influence on the expected 1:1 sex ratio either. The number of cell cycles was similar for male and female (P = 0.23) embryos when quality of the embryo (P < 0.0001) was included in the model. The results of this study indicate that, in cattle, the interval between insemination and ovulation does not influence the sex ratio of seven-day-old embryos.     

Clinical experience with flow cytometric separation of human X- and Y-chromosome bearing sperm

Numerous methods to separate human X- and Y-bearing sperm have been reported with unconfirmed separation after DNA analysis and inconsistent birth results. Successful flow cytometric separation of sperm resulting in alteration of the sex ratio of young born has been demonstrated in several animal species. Flow cytometric separation of human X- and Y-bearing sperm (MicroSort) has been confirmed after DNA analysis by fluorescence in situ hybridization (FISH). Pregnancies and births have resulted from the use of MicroSort after intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).     

Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting

Intact, viable X and Y chromosome-bearing sperm populations of the rabbit were separated according to DNA content with a flow cytometer/cell sorter. Reanalysis for DNA of an aliquot from each sorted population showed purities of 86% for X-bearing sperm and 81% for Y-bearing sperm populations. Sorted sperm were surgically inseminated into the uterus of rabbits. From does inseminated with sorted X-bearing sperm, 94% of the offspring born were females. From does inseminated with sorted Y-bearing sperm from the same ejaculates, 81% of the offspring were males. The probability of the phenotypic sex ratios differing from 50:50 were p less than 0.0003 for X-sorted sperm and p less than 0.004 for Y-sorted sperm. Thus, the phenotypic sex ratio at birth was accurately predicted from the flow-cytometrically measured proportion of X- and Y-bearing sperm used for insemination.     

Calves' sex ratio in naturally and artificially bred cattle in central Ethiopia

A study was undertaken with the objective to identify some intrinsic (genotype of the cow, estrus time and parity) and extrinsic factors (service type, service time and estrus seasons) that affect calf sex ratio in naturally and artificially bred cattle in the central highlands of Ethiopia. A total of 4657 calving events were extracted from the long-term dairy cattle genetic improvement experiment at Holetta Agricultural Research Center. Factors that affect the logit of the probability of a female calf being born were obtained by using PROC GENMODE in Statistical Analysis System. Moreover, multivariate analysis was performed using PROC LOGISTIC procedure using forward selection procedure. Accordingly, genotype of the cow, parity, estrus season, and service type had considerable influences on calf sex ratio. However, estrus time and service time did not affect calf sex ratio (χ² = 0.83 and 0.79, respectively). In Ethiopia, smallholder dairy farmers often complain that artificial insemination (AI) skewed to producing more male calves. However, our study showed that AI did not alter female-to-male calf sex ratio. On the contrary, natural mating increases the probability of female calves born (odds ratio 1.38) over AI. Heifer/cows that showed estrus and bred during the harsh seasons of the years produced more female calves than those that bred during the good seasons of the year. This strongly agreed with Trivers and Willard sex allocation theory.     

Paternity in mallards: effects of sperm quality and female sperm selection for inbreeding avoidance

Postcopulatory processes might play an important role in sexualselection. In theory, fertilization success could be controlledby females via selection of particular sperm within their reproductivetract, or it could be determined by sperm competition per se.In practice, these two mechanisms are difficult to disentangle.To assess the relative importance of both mechanisms we usedartificial insemination in combination with measurements ofsperm quality (swimming speed and motility) in mallards. Inthis species, females often lack behavioral control over copulationsand hence may use postcopulatory mechanisms to optimize theirreproductive output. One important factor affecting female fitnessmay be selection of genetically compatible males. To investigatethe influence of sperm quality and parental relatedness on paternitywe inseminated 12 groups of related females with a sperm mixturecontaining equal numbers of sperm from a brother and from anunrelated male. Paternity was independent of the relatednessof the siring male to the female but was significantly affectedby long-term sperm swimming speed and motility. No interactionbetween relatedness and sperm quality on paternity was observed.These results suggest that female mallards are not able to selectsperm on a purely genetic basis and emphasize the importanceof sperm quality in gaining paternity.     

Relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte

The aim of this study was to explore how some reproductive methodologies may affect the sex ratio. We first confirmed the association between the maturation stage of bovine oocytes at the time of in vitro fertilisation (IVF) and the sex ratio of in vitro-derived embryos. Secondly, we studied whether the time of insemination, prior to or after ovulation, could alter the sex ratio in sheep. In the first experiment, bovine oocytes were matured in vitro for 16 h; then oocytes were either fertilised in vitro immediately after extrusion of the first polar body or IVF was delayed for 8 h. The proportion of cleaving embryos and their development to the 8-cell stage was enhanced with delayed insemination. Moreover, delaying IVF produced a male-to-female sex ratio of 1.67:1.00, which was significantly different from the expected 1:1 ratio (p < 0.05), whereas more female embryos were produced when oocytes were fertilised in vitro immediately after polar body extrusion (sex ratio of 1.00:0.67; p < 0.05). In the second experiment, 380 ewes were inseminated at different times before or after ovulation, producing 537 lambs. Significant differences in the sex ratio were obtained when we compared the sex of the offspring of ewes inseminated during the 5 h preceding ovulation (more females) with those inseminated during the 5 h after ovulation (more males). Our results suggest that the differential ability of X- or Y-bearing spermatozoa to fertilise oocytes depending either on time of insemination or oocyte maturation state, may be due, at least partially, to 'intrinsic' differences in the physiological activity of X- or Y-bearing spermatozoa before fertilisation.     

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii)

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.     

Sex-specific spawning behavior and its consequences in an external fertilizer

Identifying the target of sexual selection in externally fertilizing taxa has been problematic because species in these taxa often lack sexual dimorphism. However, these species often show sex differences in spawning behavior; males spawn before females. I investigated the consequences of spawning order and time intervals between male and female spawning in two field experiments. The first involved releasing one female sea urchin's eggs and one or two males' sperm in discrete puffs from syringes; the second involved inducing males to spawn at different intervals in situ within a population of spawning females. In both, fertilization success was measured as the fraction of eggs fertilized and the paternity share of each male. The results indicate that spawning after females imposes a cost on males but only during sperm competition. Further, the optimal interval between the initiations of male and female spawning depends on degree of sperm competition, distance between males and females, and water velocity. The results show that sex differences in spawning timing of marine invertebrates can be explained on the basis of the differential costs and benefits of spawning out of synchrony with the other sex and that the result of sexual selection on external fertilizers may be behavioral rather than morphological differentiation of the sexes.