By
Glenn R. Wehner², C. Wood, A. Tague³, D. Barker³, and H. Hubert³

Northeast Missouri State University, Kirksville, MO 63501, USA

Accepted 2 August 1996

Abstract

Seventy-five spring calving Gelbvieh and Angus cows were utilized over a three year period to evaluate the use fullness of the OVATEC intravaginal probe for indicating the onset of estrus and providing the possibility to influence the sex of the off spring by choosing a breeding time in relation to critical changes in cervical mucus conductivity. Cows were randomly assigned by breed each year into one of 4 treatments: (1) probed and inseminated when impedance values declined, creating conditions expected to favor x- bearing sperm and with it an increase in the conception of females (PF); (2) probed and inseminated when impedance values were rebounding, expected to favor y-bearing sperms and with it an increase in the conception males (PB); (3) standing estrus (AI); or (4) natural service by bull (NS) Cows grazed or were fed hay from tall fescue-legume pastures. Lutalyse was used to synchronize estrus in a two-injection scheme. Vaginal probe readings were taken at first injection, second injection and every 12 hours thereafter for 6 days. Visual observations for estrus were obtained for PF, PB and AI every 12 hours postsecond injection. Rectal palpations of ovaries were obtained at standing heat in all but NS treatments. In cycling cows, probe readings increased prediction of estrus onset (P<. 10) compared to visual observations and were similar (P>. 95) to rectal palpations in all probed cows. PF cows delivered heifer calves at greater rates (P<. 025) than all other treatments whereas PB cows delivered bulls at greater rates (P<. 05) than all other treatments. Heifer to bull ratios were not different (P>. 95) for AI or NS treatments. The results of this study indicate that the potential exists for increasing female offspring conceptions utilizing cervical mucus conductivity as a gauge for insemination times.

KEY WORDS: Cattle-oestrus; Oestrus detection; Cervical micuc conductivity; Sex prediction

1. Introduction

The introduction of artificial insemination (AI) into the beef cattle industry has allowed for tremendous economic and genetic benefits. However, it is essential that the time of insemination coincide with a fertile estrus (Williamson et al., 1972; Esselmont and Bryant, 1976). A major drawback to AI use is non-reliable estrus detection (Holmann et al., 1987; Williams et al., 1981). Several researchers has shown that electrical impedance in vaginal mucus may be used to pin point proper insemination time (Edwards and Levin, 1974; Leidl and Stolla, 1976; Schams et al. , 1977; Edwards, 1980; McCaughey and Patterson, 1981; scipioni et al. , 1982). Decreasing electrical resistance values were always associated with the onset of estrus (Feldman et al., 1978; Schams et al., 1977; McCaughey and Patterson, 1981; scipioni et al., 1982; Straub et al., 1984). Furthermore, McCaughey and Patterson (1981) reported that the lowest resistance readings from excised tracts were those that exhibited the greatest follicular activity. Schams et al. (1977) reported that the lowest readings always corresponded with the luteinizing hormone (LH) peak. Straub et al. (1984) found that this change in electrical resistance of vaginal mucus was actually more accurate in determining the correct time to breed than visual observation of behavioral estrus. Major drawbacks to this method are that accurate individual cows records are essential and that there are response differences between individual animals making it necessary to establish a response curve for each animal making universal application very difficult if not impossible (Senjum, 1985).

Unpublished pilot studies conducted by the Profitable Breeding Corporation (1986 to 1988) and this laboratory indicated that vaginal-cervical mucus conductivity readings obtained with OVATEC unit appeared to be more consistent than previous units. Furthermore, patterns of response in individual animals were similar across several breeds of cattle in different herds with no treatment X operator interactions leading to possible universal application of OVATEC readings for the prediction of estrus onset in cattle. This consistence is due to the OVATEC unit's combination of electrical resistance and capacitance parameters in the calibration and sensing of ion fluxes, whereas other commercially available units utilize resistance measurements only.

Additional observations in these pilot studies as to the correct time to artificially inseminate the test animals revealed that critically timed breeding based on OVATEC readings of electrolyte fluctuations might influence the gender of resultant offspring. Inseminations performed at declining readings of 45 to a terminal decline reading of 35, on the average of 20+3h preovulation, appeared to result in a higher than expected ratio of female to male offspring. Conceptions obtained by inseminations performed on a rebounding scale of 50 to 70, occurring 10+2h preovulation, seemed to favor male offspring. The literature is lacking on the use of such timed breeding to influence gender of offspring.

The objectives to this experiment were to (1) evaluate the efficiency of the OVATEC unit to detect estrus onset in an uniform pattern among cows and (2) to evaluate the ability of the unit to indicate proper breeding time so as to influence calf gender.

2. Material and Methods

The intravaginal probe (OVATEC) was supplied by Profitable Breeding Corporation, Inc. and used according to the company's suggested procedure. The 13 cm long x 1.5 cm diameter probe was scrubbed for 1-2 minutes before each group of readings with an abrasive cleanser (i.e. Comet, Bon Ami, Ajax) to remove any oxidation and tested in a sodium chloride (.07N) test solution for calibration. The probe was cleaned and sanitized with NOVASAN, a germicidal disinfectant, by wiping with a disposable disinfectant soaked paper towel prior to and between use on each cow to avoid transfer of infection (scipioni et al., 1982). In addition, the probe was kept in a tube filled with NOVASAN between use on each cow and rinsed with hot water (490 C) prior to vaginal insertion.

Probed cows were confined in a squeezed chute and the vulva cleaned with a mild soap solution and rinsed with clean water. The lips of the vulva were spread slightly to allow sanitary insertion of the probe. Insertion was approximately at 45-degree angle upward for 15 centimeters and then straightened to a horizontal position until it bumped the cervix. The probe was the gently moved back and forth with a half turn twisting motion for 20-30 seconds to stimulate vaginal-cervical mucus flow and coat the probe. The probe was then carefully pushed to the cervix and held horizontally with slight downward pressure. A digital recording was obtained when display numbers stopped changing. A probe reading of less than 90 (the Base Estrous Reading=BER) indicated the cow was proceeding into estrous (Profitable Breeding Corporation, 1987).

In a complete randomized design, 75 mature spring calving Gelbvieh and Angus cows were assigned to one of four treatments: (1) probed and AI bred to select for heifer calves (PF). Cows bred so as to select for heifer calves were AI serviced between readings of 45-35 on the decline; (2) probed and AI bred selecting for bull calves (PB). Cows bred to have bull calves were AI serviced between 50 70 on the rise; (3) AI serviced on visual observation of standing heat (AI); or (4) natural service by bull (NS) in each of the three years of the study. The semen used was obtained and frozen from the bull utilized in the NS treatment group. Cows not settling to insemination efforts in PF, PB or AI treatment were included in the NS group. Estrus in all cows was synchronized with two 5 ml (25 mg prostaglandin F2,) Lutalyse¹ injections 10 days apart. Vaginal probe readings for PF and PB cows were obtained at first and second injection of Lutalyse and every 12 hours thereafter (0600 and 1800 hrs) until insemination. Probe readings changed at a rate of 1 to 1. 5 units per hour when the cow proceeded into a normal heat cycle, thus estimates of proper insemination times were calculated and cows reprobed at the time estimated to correspond to a proper reading for the PF and PB groups.

Table 1.

ESTRUS DETECTION BY THREE METHODS FOR ALL ARTIFICIALLY BRED COWS

Detection Method

Table 2.

OBSERVED CALF SEX FREQUENCIES AND CHI-SQUARE VALUES FOR TREATMENTS

Treatment

AI treatment cows were visually appraised every 12 hours (0600 and 1800 hrs, respectively) and inseminated upon detection of behavioral estrus. NS cows were synchronized and placed with the same fertile bull for 24 days post second injection in each of the 3 years of the study.

All cows in the PF, PB and AI treatments were rectally palpated and probed upon visual detection of estrus behavior regardless of insemination treatment to compare probe predictions of estrus to physiological estrus (rectal palpation of graffican follicle) and psychological estrus (standing heat behavior) . First service conception rate for PF, PB and AI cows averaged 80, 84 and 82%, respectively, over the three years.

Calving dates varied between February through April each year depending on breeding dates. Calving data were collected at birth for calf weight, calf identification, dam identification and sex of calf. Cows grazed or were winter fed hay from similar pastures consisting of approximately 60% Kentucky-31 tall fescue (Festuca arundinacea Schreb.) and 40% red clover (Trifolium pratense L.).

Preliminary analysis indicated no breed or year differences (P>.75) therefore, data was combined for the three years. Means for method of estrus detection (Table 1) were separated using LSD procedures (Snedecor and Cochran, 1967a). Observed frequencies for treatments were subjected to Chi Square analyses (Table 2) (Snedecor and Cochran, 1967b).

3. Results and Discussion

OVATEC readings for estrus detection in both PF and PB groups were more dependable (P<. 10) than visual observation of estrus (Table1). These findings compare favorably with those of Schams et al. (1977), Scipioni et al. (1982) and Straub et al. (1984). Method of estrus detection did not differ (P>.95) in the AI treatment groups since probable reading and rectal palpations were performed upon visual observation of normal estrus behavior. Although there was a possibility of cycling cows being missed by visual appraisal, it is also probable that cycling cows may not have exhibited any behavioral aspects. This would indicate that OVATEC readings conducted every 12 hrs following the second PGF2, injection are more reliable than 12 hr visual observations and would eliminate the technical skills necessary for rectal palpation detection of estrus. Larsson (1987) reported that the time between the peak of luteinizing hormone and ovulation averaged of 24.9 h. PF cows were bred at OVATEC readings of 45-35 on the decline and averaged 3 reading prior to service, ovulation occurred at 60 to 70 on the rise, with readings changing 1.0 to 1.5 per hour. This lead to an average preovulation insemination of 20 hours for PF cows which compares favorably with results of Larsson (1987). PB cows were bred + 8 hr of ovulation and averaged 6 readings prior to service. Furthermore, the ability of probe readings to parallel actual standing heat behavior and rectal palpation data in the AI treatment group suggests that OVATEC readings accurately reflect estrus activity consistently from cow to cow.

In a retrospective analysis of data, Steinel (1981) reported that male sex tended to be favored by 12% when cows were inseminated late in standing heat with separated semen. Whereas, earlier insemination (i.e.: closer to ovulation) resulted in a 50:50 ration. Work by Kiepe (1978) revealed similar trends. The literature is lacking concerning the effects of preselected time of insemination on sex of the resultant calf. Table 2 illustrates such a relationship (P<.005) with PF and PB treatment groups for observed postcalving sex frequencies. No such relationship was evident from the frequencies for AI or NS groups. Male:female ratios favored female calves (P<.025) in PF treatment cows, whereas B cows delivered a higher ratio (P<.05) of male calves (Table 2). Male : female ratios did not differ (P>.95) in AI nor NS treatment groups from expected values. These results reflect presumed early physiologic shifts in ion concentrations in cervical mucus related to the onset of estrus and not necessarily the psychologic changes related to hormonal shifts with concomitant changes in behavior (ie: standing heat).

Physiologic control of calf sex would seem to be most probably related to sperm capacitation, which is necessary for fertilization to occur. It has been demonstrated that secretions for the uterus and oviduct, as well as follicular fluid released at ovulation, participate in the capacitation process (Hunter and Hall, 1974; Iritani and Niva, 1977; Esbanshade and Clegg, 1980; Herz et al., 1985). Research has demonstrated that ion fluxes in these fluids control capacitation activities. Induction of acrosomal exocytosis has been mediated by agents such as calcium concentration in the uterine environment (Yanagimachi, 1975; Green, 1978; Triana et al., 1980; Parrish et al., 1988; Roldan and Harrison, 1989). Parrish, (1992) reported that the uptake of calcium ions by bovine sperm during capacitation was sequential, increasing in intracellular concentration over time. Increases in potassium ion concentrations promoted a concomitant increase in calcium uptake by bovine spermatozoa (Babcock, 1988) with the calcium uptake accelerated under alkaline conditions (Garcia-Sota et al., 1987). Tash et al., (1988) reported that phosphorylation reactions were necessary for sperm mobility and these mimicked calcium alterations of sperm mobility. Babcock (1983) revealed potassium-dependent increases in cystosolic pH stimulated metabolism and mobility of sperm cells. If spermatozoa carrying a Y chromosome capacitate earlier postinsemination due to a greater sensitivity to uterine ion concentration than those carrying an X chromosome, uterine-oviduct environment mediated sex selection could occur.

Female selection due to earlier preovulatory insemination might also be explained by this theory. Iritani and Nieva (1977) reported the bovine sperm required 3-4 hours to capacitate in vitro. Larsson (1988) concluded that large numbers of spermatozoa were stored in the uterotubal junction and this reservoir is established at two hours postinsemination in animals inseminated early in estrus. From this reservoir, spermatozoa are capacitated and released to the upper part of the oviducts. Thus, if early insemination encouraged early Y bearing sperm capacitation and then their death, only x bearing sperm would survive at ovulation. Another possibility is decapacitation of Y bearing sperm. Gibbons and Gibbons (1973), Brokaw et al., (1974) and Brokaw (1979) reported that high intrasperm levels of calcium ion lead to complete inhibition of mobility. These authors reported the effects of sequentially increasing the aqueous level of free calcium ion on sperm mobility in dismembraned models was first capacitation, followed by altered flagellar waveforms and then induced quiescence. If Y bearing sperm were more rapid in the sequential uptake of calcium described by Parrish (1992) then their immobility would lead to mobile X bearing sperm in higher concentrations at the time of fertilization and thus, influence a larger number of female offspring.

Further research is indicated to determine the mechanism of sperm selection that appears to generate a significant control on survival or presence of X chromosome bearing sperm cells following early preovulatory insemination.

Acknowledgements

The authors would like to thank Dr. David Cross and Ms. Andrea Davis for their assistance in preparation of this manuscript and Ms. Kathryn Schutter for typing the manuscript.

References

Aizinbudas, L. B. and P. 0. Dovil’tis. 1962. Electronic method for inseminating cows. Zhivotnovodstvo 11:68.

Babcock, D. F. 1988. Cross talk between voltage-dependent components of the signal transduction machinery of bovine spermatozoa. Biol. Repro. 38 (Suppl. 1):60.

Babcock, D. F., G.A. Rufo, Jr., and H. A. Lardy. 1983. Potassium- dependent increases in cytosolic pH stimulate metabolism and mobility of mammalism sperm. Proc. Natl. Acad. Sci. 80:1372.

Bedford, J. M. 1983. Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol. Reprod. 28:108.

Brokaw, C. J., R. Jossolin, and L. Bobr'ow. 1974. Calcium ion regulation of flageller beat symmetry in reactivated sea urchin spermatozoa. Biochem. Biophys. Res. Commun. 58:795.

Brokaw, C. J. 1979. Calcium-induced asymmetrical beating of triton-demembranated sea-urchin spermatozoa. J. Cell. Bio. 62:401.

Edwards, D. F. 1980. Proposed instrumentation to determine the optimum time to inseminate cattle by measurement of vaginal impedance. Med. and Biol. Eng. and Comput. 18:73.

Edwards, D. F. and R. J. Levin. 1974. An electrical method of detecting the optimum time to inseminate cattle, sheep and pigs. Vet. Res. 95:416.

Esbenshade, K. L. and E. D. Clegg. 1980. Acrosome reaction of sperm incubated in the uterus of gilts. Am. J. Vet. Res. 41:1137.

Esslemont, R. J. and M. J. Bryant. 1976. Oestrus behavior in a herd of dairy cows. Vet. Res. 99:472.

Feldman, F., E. Aisinbud, H. Schindler, and H. Broda. 1978. The electrical conductivity inside the bovine vaginal wall. Anim. Prod. 26:61.

Garcia- Soto, J., M. Gonzalez - Martinez, L. De la Torre and A. Darszon. 1987. Internal pH can regulate Ca 21 uptake and the acrosome reaction in sea urchin sperm. Dev. Biol. 120:112.

Gibbons, B. H. and I. R. Gibbons. 1973.Calcium-induced quiescence in reactivated sea urchin sperm. J. Cell. Biol. 13:3-37.

Green, D. L. 1978. The induction of the acrosome reaction in Guinea pig sperm by the divalent metal cation ionosphere A23187. J. Cell. Sci. 32:137.

Herz, Z., D. Northey, and M. Lawyer. 1985. Acrosome reaction of Bovine spermatozoa in vivo: Sites and effects of stages of the estrous cycle. Biol. Reprod. 32:1163.

Holmann, F. J., R. W. Blake, and C. R. Shermway. 1987. Economic evaluation of fourteen methods of estrus detection. J. Dairy. Sci. 70:186.

Hunter, R. H. F. and J. P. Hall. 1974. Capacitation of boar spermatozoa: Synergism between uterine and tubal environments. J. Exp. Zool. 188:203.

Iritani, A. and K . Niwa. 1977. Capacitation of bull spermatozoa and fertilization in vitro of cattle follicular ocytes matured 20 in culture. J. Reprod. Fertil. 50:119.

Johnson, L. A. 1986. Gender preselection in farm animals. In: J.

J. J. Crowley (Ed) 1986 Yearbook of Agriculture: Research for Tomorrow. U.S. Department of Agriculture, Washington D.C.

Kiepe, H., 1978. GieBener Schriftenreihe Tierzucht and Haustiergenetik. Vergleichende Untersuchungen zur Fdrsenvornutzung in Reinzucht und Kreuzung. Band 40. Hamburg: Paul Parey.

Larsson, B. 1987. Determination of ovulation by ultrasound Examination and its relation to the LH - peak in heifers. J. Vet. Med., Ser.A. 34:749.

Larsson, B. 1988. Sperm distribution and its relation to ovulation in artificially inseminated heifers. Repro Domestic Anim. 23:56.

Leidl, W. and R. Stolla. 1976. Measurement of electrical resistance of the vaginal mucus as an aid for heat detection. Theriogenology. 6:237.

McCaughey, W. J. and A. D. Patterson. 1981. Vaginal electrical resistance in cows: Measurements in isolated reproductive tracts. Vet. Res. Comm. Elsevier Sci. Publ. Co. Netherlands. 73-76.

Parrish, J. J. 1992. Changes in the plasma membranae and cytoplasm of bovine sperm during capacitation. Invited paper. American Society of Animal Science 84th Annual Meeting. Pittsburgh, Pennsylvania.

Parrish, J. J., and J. L. Susko-Parrish, M. A. Winer, and N. L. First. 1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38:1171.

Profitable Breeding Corporation.

Roldan, E. R. S. and R. A. P. Harrison, 1989. Polyphospho inositide breakdown and subsequent exocytosis in the Ca 21 /ionophore-induced acrosome reaction of mammalian spermatozoa. Biochem. J. 259:397.

Schams, D., E. Schallenberger, B. Hoffmann and H. Karg. 1977. The oestrus cycle of the cow: Hormone parameters and time relationships concerning oestrus, ovulation and electrical 23 resistance of the vaginal mucus. Acta Endocrinol. 86:180.

Scipioni, R. L., R. H. Foote, S. V. Lamb, C. E. Hall, D. H. Lein and S. J. Shin. 1982. Electronic probe measurements of cervico-vaginal mucus for detection of ovulation in dairy cows: sanitation, clinical observations and microflora. Cornell Vet. 72:269.

Senjum, N. 1988. Probe pinpoints breeding time. Dairy. Nov. 10, pp.6-8.

Snedecor, G. W. and W. G. Cochran. 1967a. Statistical Methods. Iowa State University Press, Ames. pp.271-275.

Snedecor, G. W. and W. G. Cochran, 1967b. Statistical Methods. Iowa State University Press, Ames. pp. 20-26.

Steinel, G. 198 1. Untersuchung Zur Beeinf lussung des Geschlechtes Beim Rind. Inaugural Diss., Justus-Leibig Univ., Giessen, 3 Germany.

Straub, E. A., L. A. Edgerton, and G. Heershe. 1984. Changes in electrical resistance of the vagina during estrus in heifers. Preliminary report to Animark, Inc. By Univ. of Kentucky-Lexington.

Tash, J. S., J. Krinks, J. Patel, R. L. Means, C. B. Klee and A. R. Means. 1988. Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J. Cell. Bio. 106:1625.

Tash, J. G. and A. R. Means. 1983. Cyclic adenosine 31, 51 monophosphate, calcium and protein phosphorylation in 14 flagellar mobility. Biol. Reprod. 28:75.

Triana, L. R., D. F. Bobcock, S. P. Lorton and H. A. Lardy. 1980. Release of acrosomal hyaluronidase follows increased membrane permeability to calcium in the presumptive capacitation sequence for spermatozoa of the bovine and other mammalian species. Biol. Reprod. 23:47.

Williams, W. J., D. R. Yver and T. S. Gross. 1981. Comparison of estrus detection techniques in dairy heifers. J. Dairy Sci. 64:1738.

Williamson, N. B., R. S. Morris, D. C. Blood and C. M. Cannon. 1972. A study of oestrus behavior and oestrous detection methods in a large commercial dairy herd. The relative efficiency of methods of oestrus detection. Vet. Res. 91:50.

Yanagimachi, R. 1975. Acceleration of the acrosome reaction and activation of guinea pig spermatozoa' by detergents and other reagents. Biol. Reprod. 13:519.