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Growth of the pre-ovulatory follicle, circadian distribution of ovulations and pregnancy rate after post-ovulatory inseminations in heifers
Animal Rqmducrion Science, 27 ( 1992) I 13-121 Elsevier Science Publishen B.V., Amsterdam
113
Growth of the pre-ovulatory follicle, circadian distribution of ovulations and pregnancy rate after post-ovulatory inseminations in heifers W. Klhn’ Unr wwly
o/Munrch,
Faculry qf Velerinary Medicine, W-8000 Miinchen 22, Germany
Kiiniginsrmse
12.
(Accepted 12 June 1991)
ABSTRACT Kihn, W., 1992. Gmwh of the pre-ovulalory follicle, circadian distribution of ovulations and pregnancy rate atkr post-ovulatory inseminations in heifers. Anim. Reprod. Sci.. 27: I1 3-l 2 1. The growth of pre-ovolatory follicles fmm Day -4
to -I
(Day O=f?om owlation
to 24 h after
ovulation) and thecircadiandistributionofovulations werestudiedin 41 oestrouscyclesof IS heifers by frequent ultrasound examinations. The internal diameters ofpre-ovulatory foIli& increaxd fmm I2.2f2.3mm (meanfstandarddeviation (SD))to l5.2f 1.7mmfmm961o36hpriortoovulation with an average growth rate of 1.5 mm day“. Average diameters of oestmus follicles remained between IS.2 and during the last 36 h before ovulation. Ovulations occurred randomly during the day 18/41.44%) and at sight (23/41. 56%). Relatively few ovulations were detected berween 900 and h. Nineteen nsllipmur heifers were artiticially inseminated once with frozen-thawed semen between 0 and 24 h cast-ovulation in a Iaal of 37 cvcles. Preanancv rates were 69% f9/13) in the amuo inseminated O-9 b aher ovulation and 25% (5/20) in the group inseminated aI 9-l 8 h. No pregnancy (O/4) occurred when heifers were inseminated more than I8 h atkr ovulation. The pregnancy rate decreased significantly as the time from ovulation increased (FkO.01). The outcome of the tirst insemination for each heifer was used for statistical tesring. These data demonstrate that pregnancies can few11 from post-ovulatoty inseminations in the bovine. Pregnancy rates were adequate afler inxminaIion within 9 h and low between 9 and 18 h postovulation.
(
IS.5mm
15:OO
.
_.
.
_.
INTRODUCTION
Delayed insemination relative to ovulation is known to have a deleterious effect on fertility in the bovine (Hunter, 1985). It has been assumed that insemination several hours after ovulation-nomally the day after the end of oestrus-does not result in pregnancy. This was based on the concept that ‘Present address: University of Pennsylvania, School of Veterinary Medicine, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348-1692, USA.
0 1992 Elsevier Science Publishers B.V. AU riahcs reserved 0378~320/92/$05.00
!!4
w.tsHN
retain their full viability for only 8-10 h after oulllation, whereas spermatozoa require approximately this time to perform capacitation before they are capable of fertilizing ova (Hinrichsen-Kohane et al., 1984). If suermatozoa are introduced into the female genital tract after ovulation has occurred, they reach their fertilizing ability at a time when the oocyte is already showing signs of aping and is losing viability. From the time schedule of these events, the insemination of cows several hours after ovulation does not appear to be reasonable. Contrddktory to this, it has heen reported that cows may become pregnant by insemination a substantial time after the end of oestrus, even when tbev show bloody discharge at the vulva (Autrup and Rashech, i95 1). Ovulation occurs predominantly from 24 to 36 h after the onset (Schams and Butx, 1972; Christenson et al., 1975; Schams et al., 1977; Bernard et al., 1984; Lamson; 1987; Rajamahendran ct al, 1989) or 6-12 h after the end (Marion et al., 1950) of standing oestrus, and post-oestrous baemorrbage can be observed most frequently between 48 and 72 h (Trimber8er, 1941; Weber et al., 1948) after the beginning of oestrus. It might, therefore, be supposed that insemination even the day after ovulation can lead to pregnancy. Recent studies in the equine indicate that pregnancy rates in mares may reach pre-ovulatory levels when they are bred naturally or inseminated with fresh-dilnted semen O-12 h post-ovulation, but decrease between 12 and 30 h after ovulation (Belling, 1984: Palmer, 1984; Katila et al., 1988; Woods et al., 1990). In most experiments on the influence of breeding time on feriihty in cows, pregnancy rates refer to intervals from the onset of oestrus to insemination (Robbins et al., 1978; Foote, 1979). Limited data are available taking ovulation itself as a point of reference in evaluating inmmiiuitior results. The objectives of this study were to determine: ( 1) the size of the pre-ovulatory follicle during the 4 days prior to ovulation; (2) the circadian distribution of ovulations during the day and at night; (3) the pregnancy rates of heifers inseminated at different times within the first O-24 h after ovulation. oocytes
MATERIALS AND METHODS
The studies were conducted on 11 nulliparous heifers during September 1988 to February 1989 and on eight nulliparous heifers from November 1989 to February 1990. All heifers were of the Simmentaler breed and ranged in age from 17to 29 months, weighing 380460 kg at the beginning of the experimental Period and 450-520 k8 at the end. They were maintained in groups of 3-4 animals per stall, fed twice daily with hay, once with 1.0 kg concentrates and water was supplied ad libitum. The heifers showed normal cycling activity and no signs of reproductive disorders.
EFFECTS OF Posm”“uToRv
lNSEMlNAnONs
IN A”FEn.5
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Ultrasound examinations Ovarian activity of the animals was assessed by transrectal ultrasound scanning. For sonagraphic monitoring, animals were led to an examination room and restrained in a stanchion. The following ultrasound scanners were used for transrectal aonoarams: Aloha SSD210 DX II (Helliae GmbH. W-7800 Freiburg, Germa&) with a 5.0 MHz linear array and Si&.na SLt (Sicmens AG, W-8520 Erlangen. Germauv) with a 5.0 MHz sector and a 5.0 h4Hz linear probe. Both scanners and theVthree probes were appropriate for monitoring the ovaries. A video tape recorder, video printer and Polaroid camera were connected to the ultrasound scanners for recording ovarian structures. The numbers of follicles on each ovary were counted. Internal diameters of the largest and second largest follicles, and the width and length of a corpus luteum, were measured. The pm-ovulatory follicle was defined as the follicle that became and remained the largest vesicle at least 4 days prior to oestrus and that eventually ovulated. Heifers were examined ultrasonically every 2-3 days during dioestrus and pm-oestrus. As soon as an animal exhibited signs of oestrus, the frequency of ultrasound examinations was increased up to every 3-4 h until ovulation was detected. Gesttus observationswere made by frequentlyrecording(every 2-3 days during dioestrus and up to every 3-4 h during oestrus) standing and mounting activity, and mucous vu!var discharge. Data collection for the experiments was started after the observation of at least one normal oestrous cycle. Ovulation was detincd, retrospectively. when anechoic follicular fluid of a previously large oestrous follicle had disappeared. The actual time of ovulation would have been any time within O-4 h prior to this last examination. For huther data processing, the hour of ovulation was established as the midpoint between the last ultrasound scanning, when the follicle was intact, and the subsequent monitoring, when the follicle bad collapsed. If, for instance, ovulation was detected at 4~00h and the preceding examination was done at 01:OOh, 02~30 h was taken as the time of ovulation. A total of 41 ovulations was accurately detected. Sonographic scanning of ovarian activity was continued several days after ovulation to exclude possible double ovulations and until the developing corpus luteum could be found ipsilateral to the side of the previous ovulation. Inseminations
Once ovulation was confirmed and no further follicle of more than IO mm diameter was present, heifers were mndomly assigned to be inseminated either within 18-24. 15-18.12-15.9-12.6-9.3-6 or O-3 h after ovulation. At the beginning of the experiment, post-ovulatory insemination was carried out within one 3 h interval between 12 and 24 h after ovulation. Heifers which
116
w. KAHN
did not become pregnant after this insemination were again used in one of the subsequent cycles. The following post-ovulatory insemination was then carriedout closer to ovulation, between 0 and 12 h after ovulation. Post-ovulatory inseminations were carriedout after 37 ovulations in I9 heifers. AU heifers were inseminated by a small number of operatorswith frozenthawed semen from one single ejaculate of one sire (Simmentaler) with proven fertility. Semen had been preserved in egg-yolk extender with 4% glycerol in 0.5 ml French straws. Paillettes were thawed in a water bath at 38°C for 15 s. The insemination dose was 20 million progressively motile spermatozoa. Semen was deposited into the uterine corpus approximately 2-3 cm cranial to the internal cervical OS. Pregnancies were first coatirmed sonographically between Day 25 and 30, and once more by Day 60 both rectally and sonographicafly (KZha, 1985). At the end of the experiments, 14 of 19 heifers were pregnant. !a the mezntime, 13 heifers delivered a calf and one pregnant animal was slaughtered owing to an injury.
The hypothesis that ovulation was equally likely to occur during the day or night was examined using the test for single binominal probability (Pollard, 1986). The probability of ovulation during either the day or night was hypothesized to be 0.5. The relationship between the pregnancy rate and time of insemination relative to ovulation was examined using the ,yz test for a linear trend in proportions (Snedecor and Cochran, 1980). Statistical tests were performed using only the first observation per heifer (first ovulation and first insemination) to ensure independence of the observations. A twotai1e.l significance level of 0.05 was used for all statistical tests. RESULTS
Growth of oestrous follicles The largest internal diameters of the follicles representing the follicular antrum were measured sonographically during the-4 days $or to ovulation. Pre-ovulatory follicles had an average diameter of 12.222.3 mm (mean2 standard deviation (SD) ) between 96 and 84 h prior to ovulation (Fig. 1) . Internal diameters increased from Day - 4 to Dav - 2 before ovulatioa to an extension of 15.2 5 1.7 mm. From fiour -96 io Hour - 36, the average growth rate of the pm-ovulatory follicles was I .5 mm day- ‘. Withia 36 h before ovulation, the size of oestrous follicles remained fairly unchanged. The internal diameters of ovulatory follicles were IS.52 I .5mm less t,ban I2 h priorto ovulation, with a rangeof 13-19 mm. Pregnanciesoccurred
117
Ovulation
-Day4
,
,
-ray3
-cay2
8 -%
-84
-72
-fa
Aa’
-36
,
-IBY>
-24
-I2
OIWTS
Time beforr ovulation
Growth ofthe
I)
Fig 1, internal diameters (mean+SD) of ovulatory follicles (~4 from different oestmur cycies in heifers (n= 18) during the 4 days prior to ovulation. Numbers within the diagram
represenlthe couot
ODay (~SII-IB:W): g Night (18:006:00):
10
(n)
of ultrasound measurements.
18 (44%) 23 (56%)
1: ‘= 7 $6 $5 8’ g3 22
1 0
Fig. 2. Time of ovulation
(11x41)
during the day or al night
in heifers(II= 18).
after post-ovulatory inseminations in heifers with oestrous follicles ranging from 14 to 17 mm. Time of ovulation The time of ovulation was ultrasonically determined in 41 ovulations of 18 heifers (Fig. 2). Ovulations occurred during both the day and night. During the day (06:OO-1890 II ), 18 ovulations (44%) were detected, whereas during the night there were 23 ovulations (56%). Relatively few ovulations were
w. KXHN
118
found around the middle of the day, between 09100and 12:OOh, and between 12:00 and 15:OO h. During the first cycle, ten ovulations (56%) occurred during the day and eight (44%) occurred at night. The frequency of ovulations during the day did not differ significantly from the frequency during the night (ILO. I 1, indicating that ovulation occurred randomly with regard to time of day. Pregnancy rates after post-ovulatory inseminations
A total of 37 inseminations were performed within an interval of O-24 h after ovulation in 19 heifers. In 14 of 37 post-ovulatory inseminations (38%), the heifer became prcgnanr (Fig. 3 j. Of 13 heifers which were inseminated between 0 and 9 h after ovulation, nine (69%) became pregnant. Within the interval 9- 18 h after ovulation, 5 (25%) out of 20 heifers became pregnant. No pregnancy occurred in four inseminations carried out 18-24 h after ovulation. In the meantime, 13 cows have delivered a calf full term and one was slaughtered owing to an injury. Following the first insemination, six (32%) of the I9 heifers became pregnant between 0 and 24 h after ovulation. Five (7 1%) of the seven heifers inseminated within 9 it of ovulation became pregnant. One ( 13%) of eight heifers inseminated between 9 and 18 h after ovulation became pregnant. No pregnancies occurred in the four heifers inseminated 18-24 h after ovulation. The x2 test for a linear trend in proportions showed a significant decrease in Ovulation
Fig. 3. Prqtnattcy rates of brifers (wr 19) after post-ovulatory inseminations (n=37). Combined prc~nancy rates were 69% (9/13) in heifers inseminated O-9 h post-ovulation. 25% (5/20) in the 9-18 b goup and 0% (O/4) in the IS-24 h group. Pregnancy rates following the tinI insemination per heifer showed a linear decrease with increasing time between ovulation and insemination (P ~0.01). The rater after the first insemination were as follows: S/7 (O-9 ilJ:t/b:(Y-IYh):0/4(18-24h).
EFFm-rSOF
pregnancy
wsTo”“LAmRY
lNSEMlNATlONS
,!.I HElFERs
119
rate as the time between ovulation and insemination increased
(Pt0.01). DISCUSSION
Pre-ovulatory follicles showed an increase in size from Day - 4 to Day - 2 before ovulation with a growth rate of 1.5 mm day-‘. This growth rate is at the upper range of that reported for non-ovulating dominant follicles of the first or second wave of the ocstrous cycle (Fortune et al., 1988). A growth plateau could be noticed in oestrous follicles within 36 h before ovulation. In sonographic studies with a 1 day examination interval, this plateau was less distinct, but could still be seen in many pm-ovulatory follicles (Sirois and Fortune, 1988). A similar decreased growth rate of follicles prior to ovulation was reported in mares. It was less pronounced in mares examined ultrasonically daily (Pierson and Ginther, 1985) and more obvious in mares monitored twice a day (Will et al., 1988). These results support findings that ovulation in heifers and cows can occur at any time during the day (Larsson, 1987). A diurnal pattern for the distribution of ovulations is not very distinct. Whereas in this study ovulations tended to take place less frequently around noon (from 09~00to 15:00 h) and more frequently at night, other workers found a higher incidence around noon (Schams et al., 1977). Previous observations in cows showed that oestrusrelated events occur during the whole day with a tendency to circadian patterns for some behavioural or endocrine parameters. Sexual activity, expressed as mounting activity, was recorded during all daytime periods with the incidence of mounting being highest from approximately 2O:OOto 05:OO h and relatively low between 06~00and 13:OOh (Esslemont and Bryant, 1976). In the same study, the onset of oestms appeared to be evenly distributed throughout the 24 h periods. Pm-ovulatory luteinizing hormone (LH) peaks were detected most frequently by 8 a.m. and 8 p.m. (Schams et al., 1977). It is apparent from the data reported here that pregnancy can result from post-ovulatory insemination even a substantial time after ovulation. Although the studies were limited by animal numbers, the results indicate that the fertility of cows inseminated with frozen-thawed semen within O-9 h after ovulation may be similar to that expected for insemination before ovulation. The pregnancy rate averaged 69% for inseminations 0 and 9 h post-ovulation, and was equivalent to pre-ovulatory results in heifers (Laster et al., 1972). The pregnancy rate fell to 25% (5/20) between 9 and I8 h after ovulation, but was still remarkable. In earlier studies based on rectal palpation for detection of ovulation, fertility was already decreased in cows inseminated from 6 h before to 2 h after ovulation (Trimberger, 1948). However, the results of this investigation indicate that the decline in fertility commenced later. Fertility rates for post-ovulatory insemination with frozen-thawed semen
w. KAHN
120
within O-9 h after ovulation can be expected to be equivalent to pregnancy rates for inseminations the day before ovulation. Similar results have been found in mares (Woods et al., 1990). It has been shown that fertility was high (79% and 6S%, respectively) in mares inseminated with fresh-extended semen O-6 and 6-l 2 h after ovulation, but declined between 12 and 30 h after ovulation (Katila et al., 1988, Woods et al., 1990). A comparison between the findings in the equine and the bovine provides some indication that fertilization after post-ovulatory insemination may fo!low a similar time schedule in both species. For optimum timing of routine inseminations under field conditions, it can be recommended that the breeding of cows during the second half of the day after the onset of oestrus should give maximum conception rates (Robbins et al., 1978; Foote, 1979). Following this schedule, most animals are insemin&ted several hours prior to ovulation. However, based on previous studies and the present results, it is obvious that pregnancies can occur from insemination the day following ovulation. In some cases, this will be l-2 days after oestrus, even when bloody discharge from the vulva is visible. This is coasistent with earlier investigations in which cows showing post-oestrous haemorrhage were inseminated and reported to have a pregnancy rate of 21% (Larson aad Bayley, 1955). The occasionally used practice of evaluating oestrus in cows by palpating the ovulatory follicle, and refusing insemination in the absence of an oestrous follicle, is not justified in all instances. If it is reliably known that ovulation took place within a few hours prior to aa examination, insemination can still be carried out successfully. ACKNOWLEDOEMENTS
The author wishes to thank Pamela Spencer for valuable statistical advice and her assistance in the analysis of data. REFERENCES Autrup, E. and Rasbech, N.O., 1951. Befiugtningsresulmter ved kunscig insemincring med tyresperma i den poswstrale, hamorriwgiske periodc. Nerd. Veterinaemxd., 3: 40-46. Belling. T.H., 1984. Postovulation breeding and related reproductive phenomena in the mare. Equine Pratt., 6(6): U-19. Bernard. C.. Lambert, R.D., B&land. R. and BClanger, A.. 1984. Laparoscopic investigation of the bovine ovary in the periovulatory phase of the cycle. Tberiogenology, 22: 143-l 5’). Christenson. R.K.. Echtemkamp, SE. and Laster, D.B.. 1975. Oes~rus, LH, ovulation and fertility in beefheifers. 1. Reprod. Feriil., 43: 543-546. Esslemont. R.J. and Bryanl. M.J., 1976. Oestmus behaviour in a herd ofdairy cows. Vel. Rec., 99: 472-41s. Foote, R.H., 355-358.
1979. Time
ofartificialinsemination
and fenilily
in dairy cattle. J. Dairy SC.. 62:
Fortune, J.E., Sir&. J. and Quirk, SM., 1988. The growth and differentiation of ovarian follicles during the bovine estrous cycle. Tberiogenology, 29: 95.109. Hitttichsen-Kohane, A.C., Hinrichscn, M.J. and Schill, W.-B., 1984. Molecular event: leading IO fertilizaIion-A review. Andrologir, 16: 32 I-341. Hunter, R.H.F., 1985. Fertility in cattle: basic reasons why late insemination must he avoided. Anim. Breed. Abstr., 53: 83-87. Kahn, W., 1985. Zur Ttichtigkeitsdiagnose beim Rind mittels Ultrascball. Tieraentl. Umsch., 40: 472-477. Katila, T., Koskinen, E., Kuntsi, H. and Lindebcrg. H., 1988. Fertility after postovulatory inseminations in mares. I Ith International Congress on Animal Reproduction and Artificial Insemination, Dublin, Eire, Vol. 2, p. 96. Larson, G.L. andBayley, N.D., 1955. The fertilityof inseminationsmade incowssbowingpostestrous hemorrhage. J. Dairy Sci., 38: 849. Larssott. B., 1987. Determination of ovulation by ultrasound examination and its relation to the LH-peak in heifers. J. Vet. Med. .A. 34: 749-754. Laster. D.B., Glimp, H.A. and Gregory, KE., 1972. Age and weight at puberty and conception in different bncds and brced-crosscs of heef heifers. J. Anim. Sci., 34: 1031-1036. Marion. G.B., Smith, V.R., Wiley, T.E. and Barrett, G.K., 1950. The tffm of sterile copulation on time of ovulation in dairy bdfers. J. Dairy Sci.. 33: 885-839. Palmer, E., 19R4. Factors affecting stallion semen survival and fertility. 10th lntemational Congrcsson Animal Reproduction and Artificial Insemination, Urbana-Champaign, Illinois, USA, vol. III, p. 377. Pierson, R.A. and Gintber, D.J., 1985. Ultrasonic evaluation of the preovulatory follicle in the mare. Tbetiogenology, 24: 359-368. Pollard, J.H., 1986. Hypothesis testing. In: J.H. Pollard (Editor), Numerical and Stttistical Techniques. Cambridge Univenity Press, Cambridge, p. 133. RaJamabendran, R., Robtnson, 1.. Desbottes, S. and Walton, IS.. 1989. Temporal relationships among estrus, body temperature, milk yield, progesterone and luteinizing hormone levels, endovulationindaiwcow.Tberioaenolow.31:1173-1181. Robbinr, R.K.. Sul1ivan.J.J.. Pace, M.M.. El&t, EL, Bartlett,D.E. and Press, P.J.. 1978. Timing the insemination ofheefcattle. Theriogenology, I’): 247-255. Scbams, D. ad Buts, H.D.. 1972. Zeitlicbe Fkziehungen zwiscben Brunstsymptonten. elektrischcn WiderstandsverJnderungen des Vaginalscbleims, prtlovulatoriscber Ausschiltmng dcs l.uteinisierttngshormotts und Ovulation beim Rind. Zuchtbygicne, 7: 49-56. Schams. D., Schallenherger, E., Hoffmann, JJ. and Karg, H., 1977. The oestfous cycle of the cow: hormonal pammeters and time relationships concerning oesttus, ovulation, and electri. cal reaiatanw ofthe vaginal mucus. Acta Endoctinol. (Copenhagen), 86: 180-192. Sirois. J. and Fortune, J.E., 1988. Ovarian follicular dynamics duringthe estrous cycle in heifers monitored by real-time ultrasonogmphy. Biol. Reprod., 39: 308-317. Snedecor, G.W. and Cocbran. W.G., 1980. Statistical Methods. 7th Edn. State University Press, Ames, I.4 pp. 206-208. Trimberger, G.W., I94 I. Menstruation frequency and its relation to conception in dairy cattle. J. Dairy Sci., 24: 819-823. Trimbewr, G.W.. 1948. Breeding efftciency in :iaity cattle from artilicial insemination at various intervals before and after ovttla:ion. Nebr. Agric. Exp. Sm. Res. Bull., 153: 3-26. Web& A.F., Morgan, B.B. and McNult, S.H., 1948. Clinical and postmortem observationson melmrrhagia in the virgin heifer. North Am. Vet., 29: 705-710. Will, K.. Klibn, W. and Lcidl, W., 19P8. Sonographirche Untersuchungtn ilher die pmowlatotische Follikelentaicklung hei der Stute. Dtsch. Tieraerctl. Wochenschr., 93: 363-365. Woods, J., Bergfelt. D.R. and Ginther, O.J., 1990. Enbcts of time of insemination relative to ovulation on prcgnaacy rate and embryonic-loss rate tn mares. Equine Vet. J., 22: 410-415.
113
Growth of the pre-ovulatory follicle, circadian distribution of ovulations and pregnancy rate after post-ovulatory inseminations in heifers W. Klhn’ Unr wwly
o/Munrch,
Faculry qf Velerinary Medicine, W-8000 Miinchen 22, Germany
Kiiniginsrmse
12.
(Accepted 12 June 1991)
ABSTRACT Kihn, W., 1992. Gmwh of the pre-ovulalory follicle, circadian distribution of ovulations and pregnancy rate atkr post-ovulatory inseminations in heifers. Anim. Reprod. Sci.. 27: I1 3-l 2 1. The growth of pre-ovolatory follicles fmm Day -4
to -I
(Day O=f?om owlation
to 24 h after
ovulation) and thecircadiandistributionofovulations werestudiedin 41 oestrouscyclesof IS heifers by frequent ultrasound examinations. The internal diameters ofpre-ovulatory foIli& increaxd fmm I2.2f2.3mm (meanfstandarddeviation (SD))to l5.2f 1.7mmfmm961o36hpriortoovulation with an average growth rate of 1.5 mm day“. Average diameters of oestmus follicles remained between IS.2 and during the last 36 h before ovulation. Ovulations occurred randomly during the day 18/41.44%) and at sight (23/41. 56%). Relatively few ovulations were detected berween 900 and h. Nineteen nsllipmur heifers were artiticially inseminated once with frozen-thawed semen between 0 and 24 h cast-ovulation in a Iaal of 37 cvcles. Preanancv rates were 69% f9/13) in the amuo inseminated O-9 b aher ovulation and 25% (5/20) in the group inseminated aI 9-l 8 h. No pregnancy (O/4) occurred when heifers were inseminated more than I8 h atkr ovulation. The pregnancy rate decreased significantly as the time from ovulation increased (FkO.01). The outcome of the tirst insemination for each heifer was used for statistical tesring. These data demonstrate that pregnancies can few11 from post-ovulatoty inseminations in the bovine. Pregnancy rates were adequate afler inxminaIion within 9 h and low between 9 and 18 h postovulation.
(
IS.5mm
15:OO
.
_.
.
_.
INTRODUCTION
Delayed insemination relative to ovulation is known to have a deleterious effect on fertility in the bovine (Hunter, 1985). It has been assumed that insemination several hours after ovulation-nomally the day after the end of oestrus-does not result in pregnancy. This was based on the concept that ‘Present address: University of Pennsylvania, School of Veterinary Medicine, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348-1692, USA.
0 1992 Elsevier Science Publishers B.V. AU riahcs reserved 0378~320/92/$05.00
!!4
w.tsHN
retain their full viability for only 8-10 h after oulllation, whereas spermatozoa require approximately this time to perform capacitation before they are capable of fertilizing ova (Hinrichsen-Kohane et al., 1984). If suermatozoa are introduced into the female genital tract after ovulation has occurred, they reach their fertilizing ability at a time when the oocyte is already showing signs of aping and is losing viability. From the time schedule of these events, the insemination of cows several hours after ovulation does not appear to be reasonable. Contrddktory to this, it has heen reported that cows may become pregnant by insemination a substantial time after the end of oestrus, even when tbev show bloody discharge at the vulva (Autrup and Rashech, i95 1). Ovulation occurs predominantly from 24 to 36 h after the onset (Schams and Butx, 1972; Christenson et al., 1975; Schams et al., 1977; Bernard et al., 1984; Lamson; 1987; Rajamahendran ct al, 1989) or 6-12 h after the end (Marion et al., 1950) of standing oestrus, and post-oestrous baemorrbage can be observed most frequently between 48 and 72 h (Trimber8er, 1941; Weber et al., 1948) after the beginning of oestrus. It might, therefore, be supposed that insemination even the day after ovulation can lead to pregnancy. Recent studies in the equine indicate that pregnancy rates in mares may reach pre-ovulatory levels when they are bred naturally or inseminated with fresh-dilnted semen O-12 h post-ovulation, but decrease between 12 and 30 h after ovulation (Belling, 1984: Palmer, 1984; Katila et al., 1988; Woods et al., 1990). In most experiments on the influence of breeding time on feriihty in cows, pregnancy rates refer to intervals from the onset of oestrus to insemination (Robbins et al., 1978; Foote, 1979). Limited data are available taking ovulation itself as a point of reference in evaluating inmmiiuitior results. The objectives of this study were to determine: ( 1) the size of the pre-ovulatory follicle during the 4 days prior to ovulation; (2) the circadian distribution of ovulations during the day and at night; (3) the pregnancy rates of heifers inseminated at different times within the first O-24 h after ovulation. oocytes
MATERIALS AND METHODS
The studies were conducted on 11 nulliparous heifers during September 1988 to February 1989 and on eight nulliparous heifers from November 1989 to February 1990. All heifers were of the Simmentaler breed and ranged in age from 17to 29 months, weighing 380460 kg at the beginning of the experimental Period and 450-520 k8 at the end. They were maintained in groups of 3-4 animals per stall, fed twice daily with hay, once with 1.0 kg concentrates and water was supplied ad libitum. The heifers showed normal cycling activity and no signs of reproductive disorders.
EFFECTS OF Posm”“uToRv
lNSEMlNAnONs
IN A”FEn.5
115
Ultrasound examinations Ovarian activity of the animals was assessed by transrectal ultrasound scanning. For sonagraphic monitoring, animals were led to an examination room and restrained in a stanchion. The following ultrasound scanners were used for transrectal aonoarams: Aloha SSD210 DX II (Helliae GmbH. W-7800 Freiburg, Germa&) with a 5.0 MHz linear array and Si&.na SLt (Sicmens AG, W-8520 Erlangen. Germauv) with a 5.0 MHz sector and a 5.0 h4Hz linear probe. Both scanners and theVthree probes were appropriate for monitoring the ovaries. A video tape recorder, video printer and Polaroid camera were connected to the ultrasound scanners for recording ovarian structures. The numbers of follicles on each ovary were counted. Internal diameters of the largest and second largest follicles, and the width and length of a corpus luteum, were measured. The pm-ovulatory follicle was defined as the follicle that became and remained the largest vesicle at least 4 days prior to oestrus and that eventually ovulated. Heifers were examined ultrasonically every 2-3 days during dioestrus and pm-oestrus. As soon as an animal exhibited signs of oestrus, the frequency of ultrasound examinations was increased up to every 3-4 h until ovulation was detected. Gesttus observationswere made by frequentlyrecording(every 2-3 days during dioestrus and up to every 3-4 h during oestrus) standing and mounting activity, and mucous vu!var discharge. Data collection for the experiments was started after the observation of at least one normal oestrous cycle. Ovulation was detincd, retrospectively. when anechoic follicular fluid of a previously large oestrous follicle had disappeared. The actual time of ovulation would have been any time within O-4 h prior to this last examination. For huther data processing, the hour of ovulation was established as the midpoint between the last ultrasound scanning, when the follicle was intact, and the subsequent monitoring, when the follicle bad collapsed. If, for instance, ovulation was detected at 4~00h and the preceding examination was done at 01:OOh, 02~30 h was taken as the time of ovulation. A total of 41 ovulations was accurately detected. Sonographic scanning of ovarian activity was continued several days after ovulation to exclude possible double ovulations and until the developing corpus luteum could be found ipsilateral to the side of the previous ovulation. Inseminations
Once ovulation was confirmed and no further follicle of more than IO mm diameter was present, heifers were mndomly assigned to be inseminated either within 18-24. 15-18.12-15.9-12.6-9.3-6 or O-3 h after ovulation. At the beginning of the experiment, post-ovulatory insemination was carried out within one 3 h interval between 12 and 24 h after ovulation. Heifers which
116
w. KAHN
did not become pregnant after this insemination were again used in one of the subsequent cycles. The following post-ovulatory insemination was then carriedout closer to ovulation, between 0 and 12 h after ovulation. Post-ovulatory inseminations were carriedout after 37 ovulations in I9 heifers. AU heifers were inseminated by a small number of operatorswith frozenthawed semen from one single ejaculate of one sire (Simmentaler) with proven fertility. Semen had been preserved in egg-yolk extender with 4% glycerol in 0.5 ml French straws. Paillettes were thawed in a water bath at 38°C for 15 s. The insemination dose was 20 million progressively motile spermatozoa. Semen was deposited into the uterine corpus approximately 2-3 cm cranial to the internal cervical OS. Pregnancies were first coatirmed sonographically between Day 25 and 30, and once more by Day 60 both rectally and sonographicafly (KZha, 1985). At the end of the experiments, 14 of 19 heifers were pregnant. !a the mezntime, 13 heifers delivered a calf and one pregnant animal was slaughtered owing to an injury.
The hypothesis that ovulation was equally likely to occur during the day or night was examined using the test for single binominal probability (Pollard, 1986). The probability of ovulation during either the day or night was hypothesized to be 0.5. The relationship between the pregnancy rate and time of insemination relative to ovulation was examined using the ,yz test for a linear trend in proportions (Snedecor and Cochran, 1980). Statistical tests were performed using only the first observation per heifer (first ovulation and first insemination) to ensure independence of the observations. A twotai1e.l significance level of 0.05 was used for all statistical tests. RESULTS
Growth of oestrous follicles The largest internal diameters of the follicles representing the follicular antrum were measured sonographically during the-4 days $or to ovulation. Pre-ovulatory follicles had an average diameter of 12.222.3 mm (mean2 standard deviation (SD) ) between 96 and 84 h prior to ovulation (Fig. 1) . Internal diameters increased from Day - 4 to Dav - 2 before ovulatioa to an extension of 15.2 5 1.7 mm. From fiour -96 io Hour - 36, the average growth rate of the pm-ovulatory follicles was I .5 mm day- ‘. Withia 36 h before ovulation, the size of oestrous follicles remained fairly unchanged. The internal diameters of ovulatory follicles were IS.52 I .5mm less t,ban I2 h priorto ovulation, with a rangeof 13-19 mm. Pregnanciesoccurred
117
Ovulation
-Day4
,
,
-ray3
-cay2
8 -%
-84
-72
-fa
Aa’
-36
,
-IBY>
-24
-I2
OIWTS
Time beforr ovulation
Growth ofthe
I)
Fig 1, internal diameters (mean+SD) of ovulatory follicles (~4 from different oestmur cycies in heifers (n= 18) during the 4 days prior to ovulation. Numbers within the diagram
represenlthe couot
ODay (~SII-IB:W): g Night (18:006:00):
10
(n)
of ultrasound measurements.
18 (44%) 23 (56%)
1: ‘= 7 $6 $5 8’ g3 22
1 0
Fig. 2. Time of ovulation
(11x41)
during the day or al night
in heifers(II= 18).
after post-ovulatory inseminations in heifers with oestrous follicles ranging from 14 to 17 mm. Time of ovulation The time of ovulation was ultrasonically determined in 41 ovulations of 18 heifers (Fig. 2). Ovulations occurred during both the day and night. During the day (06:OO-1890 II ), 18 ovulations (44%) were detected, whereas during the night there were 23 ovulations (56%). Relatively few ovulations were
w. KXHN
118
found around the middle of the day, between 09100and 12:OOh, and between 12:00 and 15:OO h. During the first cycle, ten ovulations (56%) occurred during the day and eight (44%) occurred at night. The frequency of ovulations during the day did not differ significantly from the frequency during the night (ILO. I 1, indicating that ovulation occurred randomly with regard to time of day. Pregnancy rates after post-ovulatory inseminations
A total of 37 inseminations were performed within an interval of O-24 h after ovulation in 19 heifers. In 14 of 37 post-ovulatory inseminations (38%), the heifer became prcgnanr (Fig. 3 j. Of 13 heifers which were inseminated between 0 and 9 h after ovulation, nine (69%) became pregnant. Within the interval 9- 18 h after ovulation, 5 (25%) out of 20 heifers became pregnant. No pregnancy occurred in four inseminations carried out 18-24 h after ovulation. In the meantime, 13 cows have delivered a calf full term and one was slaughtered owing to an injury. Following the first insemination, six (32%) of the I9 heifers became pregnant between 0 and 24 h after ovulation. Five (7 1%) of the seven heifers inseminated within 9 it of ovulation became pregnant. One ( 13%) of eight heifers inseminated between 9 and 18 h after ovulation became pregnant. No pregnancies occurred in the four heifers inseminated 18-24 h after ovulation. The x2 test for a linear trend in proportions showed a significant decrease in Ovulation
Fig. 3. Prqtnattcy rates of brifers (wr 19) after post-ovulatory inseminations (n=37). Combined prc~nancy rates were 69% (9/13) in heifers inseminated O-9 h post-ovulation. 25% (5/20) in the 9-18 b goup and 0% (O/4) in the IS-24 h group. Pregnancy rates following the tinI insemination per heifer showed a linear decrease with increasing time between ovulation and insemination (P ~0.01). The rater after the first insemination were as follows: S/7 (O-9 ilJ:t/b:(Y-IYh):0/4(18-24h).
EFFm-rSOF
pregnancy
wsTo”“LAmRY
lNSEMlNATlONS
,!.I HElFERs
119
rate as the time between ovulation and insemination increased
(Pt0.01). DISCUSSION
Pre-ovulatory follicles showed an increase in size from Day - 4 to Day - 2 before ovulation with a growth rate of 1.5 mm day-‘. This growth rate is at the upper range of that reported for non-ovulating dominant follicles of the first or second wave of the ocstrous cycle (Fortune et al., 1988). A growth plateau could be noticed in oestrous follicles within 36 h before ovulation. In sonographic studies with a 1 day examination interval, this plateau was less distinct, but could still be seen in many pm-ovulatory follicles (Sirois and Fortune, 1988). A similar decreased growth rate of follicles prior to ovulation was reported in mares. It was less pronounced in mares examined ultrasonically daily (Pierson and Ginther, 1985) and more obvious in mares monitored twice a day (Will et al., 1988). These results support findings that ovulation in heifers and cows can occur at any time during the day (Larsson, 1987). A diurnal pattern for the distribution of ovulations is not very distinct. Whereas in this study ovulations tended to take place less frequently around noon (from 09~00to 15:00 h) and more frequently at night, other workers found a higher incidence around noon (Schams et al., 1977). Previous observations in cows showed that oestrusrelated events occur during the whole day with a tendency to circadian patterns for some behavioural or endocrine parameters. Sexual activity, expressed as mounting activity, was recorded during all daytime periods with the incidence of mounting being highest from approximately 2O:OOto 05:OO h and relatively low between 06~00and 13:OOh (Esslemont and Bryant, 1976). In the same study, the onset of oestms appeared to be evenly distributed throughout the 24 h periods. Pm-ovulatory luteinizing hormone (LH) peaks were detected most frequently by 8 a.m. and 8 p.m. (Schams et al., 1977). It is apparent from the data reported here that pregnancy can result from post-ovulatory insemination even a substantial time after ovulation. Although the studies were limited by animal numbers, the results indicate that the fertility of cows inseminated with frozen-thawed semen within O-9 h after ovulation may be similar to that expected for insemination before ovulation. The pregnancy rate averaged 69% for inseminations 0 and 9 h post-ovulation, and was equivalent to pre-ovulatory results in heifers (Laster et al., 1972). The pregnancy rate fell to 25% (5/20) between 9 and I8 h after ovulation, but was still remarkable. In earlier studies based on rectal palpation for detection of ovulation, fertility was already decreased in cows inseminated from 6 h before to 2 h after ovulation (Trimberger, 1948). However, the results of this investigation indicate that the decline in fertility commenced later. Fertility rates for post-ovulatory insemination with frozen-thawed semen
w. KAHN
120
within O-9 h after ovulation can be expected to be equivalent to pregnancy rates for inseminations the day before ovulation. Similar results have been found in mares (Woods et al., 1990). It has been shown that fertility was high (79% and 6S%, respectively) in mares inseminated with fresh-extended semen O-6 and 6-l 2 h after ovulation, but declined between 12 and 30 h after ovulation (Katila et al., 1988, Woods et al., 1990). A comparison between the findings in the equine and the bovine provides some indication that fertilization after post-ovulatory insemination may fo!low a similar time schedule in both species. For optimum timing of routine inseminations under field conditions, it can be recommended that the breeding of cows during the second half of the day after the onset of oestrus should give maximum conception rates (Robbins et al., 1978; Foote, 1979). Following this schedule, most animals are insemin&ted several hours prior to ovulation. However, based on previous studies and the present results, it is obvious that pregnancies can occur from insemination the day following ovulation. In some cases, this will be l-2 days after oestrus, even when bloody discharge from the vulva is visible. This is coasistent with earlier investigations in which cows showing post-oestrous haemorrhage were inseminated and reported to have a pregnancy rate of 21% (Larson aad Bayley, 1955). The occasionally used practice of evaluating oestrus in cows by palpating the ovulatory follicle, and refusing insemination in the absence of an oestrous follicle, is not justified in all instances. If it is reliably known that ovulation took place within a few hours prior to aa examination, insemination can still be carried out successfully. ACKNOWLEDOEMENTS
The author wishes to thank Pamela Spencer for valuable statistical advice and her assistance in the analysis of data. REFERENCES Autrup, E. and Rasbech, N.O., 1951. Befiugtningsresulmter ved kunscig insemincring med tyresperma i den poswstrale, hamorriwgiske periodc. Nerd. Veterinaemxd., 3: 40-46. Belling. T.H., 1984. Postovulation breeding and related reproductive phenomena in the mare. Equine Pratt., 6(6): U-19. Bernard. C.. Lambert, R.D., B&land. R. and BClanger, A.. 1984. Laparoscopic investigation of the bovine ovary in the periovulatory phase of the cycle. Tberiogenology, 22: 143-l 5’). Christenson. R.K.. Echtemkamp, SE. and Laster, D.B.. 1975. Oes~rus, LH, ovulation and fertility in beefheifers. 1. Reprod. Feriil., 43: 543-546. Esslemont. R.J. and Bryanl. M.J., 1976. Oestmus behaviour in a herd ofdairy cows. Vel. Rec., 99: 472-41s. Foote, R.H., 355-358.
1979. Time
ofartificialinsemination
and fenilily
in dairy cattle. J. Dairy SC.. 62:
Fortune, J.E., Sir&. J. and Quirk, SM., 1988. The growth and differentiation of ovarian follicles during the bovine estrous cycle. Tberiogenology, 29: 95.109. Hitttichsen-Kohane, A.C., Hinrichscn, M.J. and Schill, W.-B., 1984. Molecular event: leading IO fertilizaIion-A review. Andrologir, 16: 32 I-341. Hunter, R.H.F., 1985. Fertility in cattle: basic reasons why late insemination must he avoided. Anim. Breed. Abstr., 53: 83-87. Kahn, W., 1985. Zur Ttichtigkeitsdiagnose beim Rind mittels Ultrascball. Tieraentl. Umsch., 40: 472-477. Katila, T., Koskinen, E., Kuntsi, H. and Lindebcrg. H., 1988. Fertility after postovulatory inseminations in mares. I Ith International Congress on Animal Reproduction and Artificial Insemination, Dublin, Eire, Vol. 2, p. 96. Larson, G.L. andBayley, N.D., 1955. The fertilityof inseminationsmade incowssbowingpostestrous hemorrhage. J. Dairy Sci., 38: 849. Larssott. B., 1987. Determination of ovulation by ultrasound examination and its relation to the LH-peak in heifers. J. Vet. Med. .A. 34: 749-754. Laster. D.B., Glimp, H.A. and Gregory, KE., 1972. Age and weight at puberty and conception in different bncds and brced-crosscs of heef heifers. J. Anim. Sci., 34: 1031-1036. Marion. G.B., Smith, V.R., Wiley, T.E. and Barrett, G.K., 1950. The tffm of sterile copulation on time of ovulation in dairy bdfers. J. Dairy Sci.. 33: 885-839. Palmer, E., 19R4. Factors affecting stallion semen survival and fertility. 10th lntemational Congrcsson Animal Reproduction and Artificial Insemination, Urbana-Champaign, Illinois, USA, vol. III, p. 377. Pierson, R.A. and Gintber, D.J., 1985. Ultrasonic evaluation of the preovulatory follicle in the mare. Tbetiogenology, 24: 359-368. Pollard, J.H., 1986. Hypothesis testing. In: J.H. Pollard (Editor), Numerical and Stttistical Techniques. Cambridge Univenity Press, Cambridge, p. 133. RaJamabendran, R., Robtnson, 1.. Desbottes, S. and Walton, IS.. 1989. Temporal relationships among estrus, body temperature, milk yield, progesterone and luteinizing hormone levels, endovulationindaiwcow.Tberioaenolow.31:1173-1181. Robbinr, R.K.. Sul1ivan.J.J.. Pace, M.M.. El&t, EL, Bartlett,D.E. and Press, P.J.. 1978. Timing the insemination ofheefcattle. Theriogenology, I’): 247-255. Scbams, D. ad Buts, H.D.. 1972. Zeitlicbe Fkziehungen zwiscben Brunstsymptonten. elektrischcn WiderstandsverJnderungen des Vaginalscbleims, prtlovulatoriscber Ausschiltmng dcs l.uteinisierttngshormotts und Ovulation beim Rind. Zuchtbygicne, 7: 49-56. Schams. D., Schallenherger, E., Hoffmann, JJ. and Karg, H., 1977. The oestfous cycle of the cow: hormonal pammeters and time relationships concerning oesttus, ovulation, and electri. cal reaiatanw ofthe vaginal mucus. Acta Endoctinol. (Copenhagen), 86: 180-192. Sirois. J. and Fortune, J.E., 1988. Ovarian follicular dynamics duringthe estrous cycle in heifers monitored by real-time ultrasonogmphy. Biol. Reprod., 39: 308-317. Snedecor, G.W. and Cocbran. W.G., 1980. Statistical Methods. 7th Edn. State University Press, Ames, I.4 pp. 206-208. Trimberger, G.W., I94 I. Menstruation frequency and its relation to conception in dairy cattle. J. Dairy Sci., 24: 819-823. Trimbewr, G.W.. 1948. Breeding efftciency in :iaity cattle from artilicial insemination at various intervals before and after ovttla:ion. Nebr. Agric. Exp. Sm. Res. Bull., 153: 3-26. Web& A.F., Morgan, B.B. and McNult, S.H., 1948. Clinical and postmortem observationson melmrrhagia in the virgin heifer. North Am. Vet., 29: 705-710. Will, K.. Klibn, W. and Lcidl, W., 19P8. Sonographirche Untersuchungtn ilher die pmowlatotische Follikelentaicklung hei der Stute. Dtsch. Tieraerctl. Wochenschr., 93: 363-365. Woods, J., Bergfelt. D.R. and Ginther, O.J., 1990. Enbcts of time of insemination relative to ovulation on prcgnaacy rate and embryonic-loss rate tn mares. Equine Vet. J., 22: 410-415.