Effect of insemination with doses of 2 or 15 millionfrozen-thawed spermatozoa and semen deposition

site on pregnancy rate in dairy cows

Magnus Anderssona,*, Juhani Taponena,Erkki Koskinena, Merja Dahlbomb

aDepartment of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, Saari Unit,

University of Helsinki, Pohjoinen pikatie 800, FI-04920 Saarentaus, FinlandbAI Cooperative Sisamaan Jalostus, Hollola, Finland

Received 25 June 2003; received in revised form 18 August 2003; accepted 4 September 2003

Abstract

The effects of low-dose artificial insemination (AI) on pregnancy rates have seldom been studied in

lactating dairy cows. We evaluated the pregnancy results after AI with doses of 2 and 15 million

frozen-thawed spermatozoa and the effect of semen deposition in lactating dairy cows. A total of 284

first inseminations with 2 million spermatozoa and 312 first inseminations with 15 million

spermatozoa were performed on 480 dairy farms. Low-dose inseminations (2 million spermatozoa)

under field conditions in commercial dairy herds, without estrus synchronization, generally resulted

in significantly reduced pregnancy rates compared with normal doses (15 million spermatozoa). The

bull � technician effect on fertility was statistically significant. This finding indicates that there is a

high variability in fertility among bulls using 2 million spermatozoa per dose. The semen deposition

site did not influence pregnancy rates. It is concluded that a dose of 2 million frozen-thawed

spermatozoa is probably too low for most bulls to achieve acceptable pregnancy rates in dairy cows.

# 2003 Elsevier Inc. All rights reserved.

Keywords: Artificial insemination; Insemination technique; Low-dose insemination; Pregnancy rate

1. Introduction

Gender selection based on flow-cytometric separation of semen into X and Y chromo-

some-bearing spermatozoa [1] and deep artificial insemination (AI) techniques [2] will

continue to have an increasing impact on milk and meat production efficiency. Dairy cows

Theriogenology 61 (2004) 1583–1588

* Corresponding author. Tel.: þ358-19-5295301; fax: þ358-19-6851181.

E-mail address: magnus.andersson@helsinki.fi (M. Andersson).

0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2003.09.006

with high genetic value should usually produce female offspring, while cows with low

value should produce male beef-crossbred calves with high meat quality and good growth

performance. Several studies on AI using low-dose frozen-thawed and low-dose sexed

semen have been undertaken in heifers and beef cows [3,4]. However, few trial results on

inseminations with low-dose (0.5–2 million spermatozoa per dose) frozen-thawed semen

in lactating dairy cows have been published [5]. A decrease from 15 million (normal dose)

to 2 million (the commercially available dose of frozen-thawed sexed sperm for heifers and

cows) frozen-thawed spermatozoa per insemination is believed to cause a drop in

pregnancy rates. The magnitude of this drop is of economic interest to farmers and AI

companies.

The aim of the present study was to evaluate the pregnancy rate in dairy cows

inseminated with 2 million spermatozoa compared with 15 million spermatozoa using

normal or deep insemination techniques. The results will be used in decision making if sex

selection of spermatozoa will be carried out in the future by AI companies in Finland.

2. Material and methods

For this experiment, semen was collected via artificial vagina from six randomly chosen

Ayrshire bulls, 14–16 months of age, producing semen of acceptable quality at the same

bull station. After determining the sperm concentration with a photometer and after

subjective evaluation of progressive motility, one ejaculate from each bull was split and

extended into two semen concentrations: 8 and 60 million spermatozoa/ml. The extended

semen was packaged in 0.25 ml French straws; thus, the insemination doses were 2 and 15

million spermatozoa per straw. The semen was frozen in a Tris-based extender with a final

glycerol concentration of 6%. This procedure produced a total of 12 semen batches with an

excess number of straws for study purposes. The accuracy of the semen dose for each

freezing operation was rechecked post-thaw using a counting chamber.

A total of 12 selected AI technicians performed the inseminations. The technicians were

divided into two groups of six: N-technicians (N: normal inseminations) performing all

inseminations into the uterine body and the D-technicians (D: deep inseminations), skilled

in embryo transfer, who performed all inseminations into the middle of one uterine horn.

The inseminations of the six D-technicians were further divided into two groups: those

where the ovaries were palpated to detect a possible ovulatory follicle or where no

palpation was performed. Approximately half of these AIs were performed without ovarian

palpation (inseminations in either uterine horn) and the other half after palpation into the

horn ipsilateral to the side of the probable impending ovulation (inseminations in the

ipsilateral uterine horn). When the AIs were performed without ovarian palpation, every

second deep insemination was performed in the right and every second in the left uterine

horn.

Each technician received two batches (2 million spermatozoa per straw and 15 million

spermatozoa per straw) of semen from one bull. The two semen batches were equally

distributed to one N- and to one D-technician. The trial was blinded, i.e. neither the

technicians nor the farmers knew which animals were inseminated with the normal dose

and which with the low dose.

1584 M. Andersson et al. / Theriogenology 61 (2004) 1583–1588

The N-technicians used ordinary insemination guns and sheaths, and the D-technicians

used embryo transfer pistolets with side-opening IMV blue sheaths. A total of 596 single-

dose AIs into Ayrshire cows in 480 commercial dairy herds were performed in this

experiment. No estrus synchronization was performed and only the first inseminations

were included. The inseminations were performed between March and June 2002.

If not reinseminated, pregnancy diagnosis was performed approximately 3 months after

the insemination with rectal palpation by a qualified AI technician.

Statistical analysis was carried out with analysis of least squares (LS) [6]. The

insemination dose, the site of deposition and the bull � technician combination served

to explain the variation in pregnancy. The differences in pregnancy rates between the

insemination doses and the site of deposition were analyzed with the chi-square test in

Table 1 and were considered significant at P < 0:05.

3. Results

The mean pregnancy rate of the inseminations with 15 million spermatozoa was 44.9%

and with 2 million spermatozoa 31.3%. Thereby the fertility of the inseminations with 2

million spermatozoa was 69.7% of the fertility of the inseminations with 15 million

spermatozoa. Using 2 million spermatozoa per straw the pregnancy rate of all deep

inseminations was 31.7% compared to the pregnancy rate of 31.1% in uterine body

inseminations.

In the least-squares analysis, the effects of insemination dose (P ¼ 0:009) and the effect

of bull � technician (P ¼ 0:002) were statistically significant. The LS mean pregnancy

rates were 26.7, 35.0, 35.4, 36.9, 49.7, and 52.7% for the six bull � technician combina-

tions. There was no statistically significant effect of insemination site on pregnancy rate.

The effects of semen dose and semen deposition on pregnancy rates are summarized in

Table 1. The pregnancy results were significantly more favorable with doses of 15 million

spermatozoa than with 2 million spermatozoa, whether the semen was deposited into the

uterine body (P < 0:01) or either uterine horn (P < 0:05). In contrast, when insemination

was performed into the middle of the ipsilateral uterine horn, no significant differences

(P ¼ 0:69) in pregnancy rates were detected between doses of 2 or 15 million spermatozoa.

Semen deposition did not appear to affect pregnancy results when 15 million sperma-

tozoa were used (P ¼ 0:83). With 2 million spermatozoa, the pregnancy results were

Table 1

Ninety-day pregnancy rates and distribution of inseminations of conventional (uterine body) and deep

inseminations with either 2 or 15 million spermatozoa

Insemination site 2 � 106 spermatozoa 15 � 106 spermatozoa P

N Pregnancy rate (%) N Pregnancy rate (%)

Uterine body 164 31.1 181 46.4 <0.01

Either uterine horn 63 25.4 67 43.3 <0.05

Ipsilateral uterine horn 57 38.6 64 42.2 n.s.

Total 284 31.3 312 44.9

M. Andersson et al. / Theriogenology 61 (2004) 1583–1588 1585

numerically, although not significantly (P ¼ 0:27), better when the semen was deposited

into the horn ipsilateral to the side of the probable impending ovulation (Table 1).

The effects of bull and/or AI technician on pregnancy rate, using 2 or 15 million

spermatozoa in the insemination dose, are summarized in Table 2. No statistics were

performed on bull or technician effect alone, due to the experimental design, in which the

effects of bull and technician could not be distinguished.

4. Discussion

The present study indicates that low-dose inseminations (2 million spermatozoa) under

field conditions in commercial dairy herds, without estrus synchronizations in lactating

cows, generally result in a significantly reduced pregnancy rate compared with normal

doses (15 million spermatozoa). The insemination site did not significantly influence the

pregnancy rate, which was expected based on earlier studies [7]. The bull � technician and

the insemination dose had statistically significant effects on fertility. This finding indicates

that there is a high variability in fertility among bulls using 2 million spermatozoa per dose.

Inseminations in either horn, without ovarian palpation, gave a lower (not significant)

pregnancy rates than inseminations in the uterine body with doses of 2 million sperma-

tozoa. The most favorable (not significant) pregnancy rates were achieved after preovu-

latory follicle palpation and deep cornual insemination into the ipsilateral horn. Thus, the

migration of spermatozoa from the contralateral horn through the oviduct and abdominal

cavity into the ipsilateral oviduct appears to be of diminutive importance compared with

sperm transport within the uterus from the uterine body to the fertilization site in cows. The

effect of semen deposition in cattle was excellently reviewed by Lopez-Gatius [8]. Higher

pregnancy rates were recorded when the inseminate was deposited into the uterine horn

ipsilateral to the side of the impending ovulation than for contralateral or uterine body

Table 2

Effects of bull and AI technician on pregnancy rate using 2 and 15 million spermatozoa in the insemination dose

Bull Technician 2 � 106 spermatozoa 15 � 106 spermatozoa Difference in

pregnancy rate (%)N Pregnancy

rate (%)

N Pregnancy

rate (%)

A D1 30 26.7 32 34.8 �17.1

A N1 27 18.5 29 51.7 �33.2

B D2 21 33.3 21 38.1 �4.8

B N2 44 15.9 44 31.8 �15.9

C D3 19 15.8 20 40.0 �24.2

C N3 37 40.5 36 38.9 1.6

D D4 18 44.4 20 50.0 �5.6

D N4 20 50.0 22 68.2 �18.2

E D5 23 30.4 24 37.5 �7.1

E N5 21 23.8 23 56.5 �32.7

F D6 9 55.6 14 50.0 5.6

F N6 15 60.0 27 48.1 11.9

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inseminations [9–12]. However, Hawk and Tanabe [13] and Momont et al. [14] found no

differences in the fertilization or pregnancy rates respectively, when comparing uterine

body and unicornual insemination ipsilateral to the side of the impending ovulation. These

contradictory findings indicate that further, more extensive insemination trials are needed.

In a study by Foote and Kaproth [15], it was shown that under favorable conditions, total

sperm counts can be reduced to 10 million spermatozoa with a reduction in non-return rates

for most bulls of about 1 percentage point from the maximum. In our study, the use of 2

million spermatozoa was sufficient for only one of the six bulls, but clearly too low a dose

in four bulls. These results show that in most bulls a higher number of spermatozoa than 2

million is needed. This finding confirms the results of Den Daas et al. [5] that 95% of the

maximal conception rate is achieved in different bulls at a range of 2–17 million

spermatozoa. A thorough follow-up of results from individual bulls with low-dose sexed

semen is required. We conclude that a dose of 2 million frozen-thawed spermatozoa is

probably too low for most bulls to get acceptable pregnancy rates in dairy cows.

Acknowledgements

We thank the AI technicians and the staff of the AI cooperative for performing the field

work and Mrs. Raili Makipaa for the laboratory examinations, including collecting of the

field data. The support of all attending farmers is highly appreciated.

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