Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2005
Volume 8
Issue 4
Topic:
Veterinary Medicine
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Niżański W. 2005. COMPARISONS OF RESULTS OF INTRAVAGINAL AND INTRAUTERINE INSEMINATION OF BITCHES WITH FROZEN–THAWED SEMEN, EJPAU 8(4), #12.
Available Online: http://www.ejpau.media.pl/volume8/issue4/art-12.html

COMPARISONS OF RESULTS OF INTRAVAGINAL AND INTRAUTERINE INSEMINATION OF BITCHES WITH FROZEN–THAWED SEMEN

Wojciech Niżański
Department and Clinic of Obstetrics, Ruminant Diseases and Animal Health Care, Wrocław University of Environmental and Life Sciences, Poland

 

ABSTRACT

The aim of the study was to compare fertility data obtained from bitches after vaginal vs. transcervical intrauterine endoscopic insemination with frozen-thawed semen. The experiment was carried out on 34 bitches divided into two groups. Vaginal insemination using bovine infusion pipette was performed in the group 1 of bitches. Transcervical insemination of semen direct into uterine lumen after visualisation of cervical portion was performed in the group 2 of bitches. Semen of 14 stud dogs was used in the study. Whelping rates in group 1 and 2 were 27.8% and 68.7%, respectively (p<0.05). Mean litter sizes at birth/mean litter sizes at weaning were significantly higher (p<0.05) in group 2 of bitches (4.9 ±1.7/4.6 ±1.7) than in group 1 of bitches (3.0 ±1.2/2.6 ±0.9). The present study showed successful results of trancervival endoscopic technique of insemination of bitches with frozen-thawed semen. Fertility and fecundity indices obtained in this study are encouraging and make opportunity for widen use of frozen-thawed dog semen for insemination of bitches in routine veterinary practice.

Key words: bitch, dog, insemination, semen, cryopreservation.

INTRODUCTION

Cryopreservation procedures exert a deleterious effect on the quality of semen [9, 19]. Successive stages of cryopreservation i.e. extension of semen, cooling, equillibration, freezing and thawing effect in significant decrease of progressive motility, acrosome integrity, longevity and proportion of live spermatozoa [9, 10, 11, 19]. It is generally assumed that the ability of frozen-thawed dog spermatozoa to pass cervical barrier in genital tract of the bitch is impaired [8].

Several techniques of uterine deposition of cryopreserved dog semen were described [1, 2, 18, 21]. Wilson [21, 22] performed artificial insemination (AI) of bitches with use of endoscope introduced into cranial part of vagina. Visualisation of vaginal portion of uterine cervix allowed for introduction of catheter directly into cervical os. Clinical data concerning comparisons between results of vaginal and endoscopic transcervical AI of bitches with frozen-thawed semen are scarce [8, 15].

The aim of the study was to compare fertility data obtained in bitches after vaginal vs. transcervival intrauterine endoscopic insemination with frozen-thawed semen.

MATERIAL AND METHODS

The study was carried out on 34 bitches aged 2.5 to 7 years and 14 stud dogs aged 3 to 8 years of different breeds. Bitches were randomly assigned to two treatment groups. In the group 1 (18 females) bitches were inseminated into cranial vagina using bovine infusion pipette (length 22-45 cm, diameter 4 mm) according to previously described [14] routine procedure. In group 2 (16 females) bitches were inseminated using endoscopic equipment consisting of 30° rigid endoscope of diameter 3 mm with a outer sheath of diameter 4 mm (Olympus, Japan) and teflon 8-French gauge urinary catheter (Cook, England). The tip of the endoscope was introduced into cranial vagina i.e. within the region of paracervix. The vaginal portion of uterine cervix was visualised and urinary catheter was introduced into cervical os in cranio-dorsal direction.

Artificial inseminations were performed at day 4 and 6 after luteinizing hormone (LH) peak. The LH peak i.e. “zero” day of oestrus cycle was detected on the basis of measurement of plasma progesterone concentration by radioimmunoassay (Pregesterone Coat-a-Count kit, Diagnostic Products Corporation, Los Angeles, CA, USA). Semen was collected from stud dogs of proven fertility. Progressive motility was evaluated using Computer Assisted Sperm Analyser (IVOS ver. 12.2l, Hamilton-Thorne Bioscences, Beverly, MA, USA), morphology of spermatozoa was assessed on slides after fixation and staining of smears with Giemsa stain [9, 10]. Concentration of sperm cells per ml was estimated in Thom chamber. Percentage of live spermatozoa was evaluated using fluorescent staining SYBR-14/propidium iodide (LIVE/DEAD® Sperm Viability Kit, Molecular Probes, Eugen, OR, USA) and FACSCalibur (Becton Dickinson, Mountain View, CA, USA) flow cytometer. Cryopreservation procedures were performed using the technique of freezing in 0.5 ml straws (IMV Technologies, L’Aigle, France) described by Niżański [9]. Each straw contained 100 x106 spermatozoa. Three straws were thawed at 70°C for 5 sec. before each insemination. Semen (1.5 ml) was mixed at 33°C with 2.5 ml of thawed autologous prostatic fluid.

Age, duration of proestrus, duration of oestrus, concentration of progesterone at first AI in two groups of bitches and quality of semen used for AI of bitches in group 1 and 2 were compared using ANOVA. Fisher’s-exact test was used to compare differences in whelping rates between groups. Litter size at birth and at weaning were compared between groups using Kruskal-Wallis test. Differences were considered to be statistically significant when p<0.05. Statistical analysis was performed using Analyse-it Software 1.71 (Clinical Laboratory Statistics).

RESULTS

Age, duration of proestrus, oestrus and concentration of progesterone at first AI (Table 1) did not differ between group 1 and 2 of bitches (ANOVA, p>0.05). There were not significant differences in motility, spermatozoal morphology, acrosome integrity and proportion of live spermatozoa (Table 2) between insemination doses used for AI in group 1 and 2 of bitches (ANOVA, p>0.05). Whelping rates in group 1 and 2 were 27.8% and 68.7%, respectively. Whelping rate obtained in group 1 was significantly lower (p=0.039, Fisher’s exact test) than in group 2 (Table 3). Litter sizes at birth and at weaning were significantly higher (p<0.05, Kruskal–Wallis test) in group 2, i.e. in bitches inseminated intrauterine using endoscope, than in group 1 (Table 4, Figures 1 and 2).

Table 1. Age, duration of proestrus, duration of oestrus and plasma progesterone (P4) concentration at the time of first insemination for the 2 treatment groups of bitches. Mean ±SD (range)

Treatment
groups

Group 1
intravaginal insemination
using infusion pipette

Group 2
intrauterine insemination
using endoscope

Age (years) a

4.1 ± 1.2
(2.5-7)

4.4 ± 0.9
(3-6)

Duration of proestrus (days) a

7.8 ± 2.1
(5-12)

8.3 ± 1.8
(6-12)

Duration of oestrus (days) a

8.6 ± 1.9
(6-12)

9.1 ± 1.5
(7-12)

P4 on day of first AI (nmol l-1) a

22.9 ± 4.8
(15.3-32.8)

20.1 ± 4.5
(13.4-30.8)

a values in rows did not differ significantly p>0.05

Table 2. Characteristics of frozen–thawed semen used for artificial insemination in the 2 treatment groups of bitches (Mean ±SD)

Treatment
groups

Group 1
intravaginal insemination
using infusion pipette

Group 2
intrauterine insemination
using endoscope

Total number of spermatozoa per insemination (x 106) a

297.9 ± 21.3

299.1 ± 18.0

Sperm motility (%) a

60.2 ± 9.5

63.1 ± 8.2

Morphologically normal spermatozoa (%) a

60.7 ± 5.2

62.0 ± 5.1

Morphologically normal acrosomes (%) a

69.4 ± 3.1

69.7 ± 2.8

Live spermatozoa (%) a

58.8 ± 7.1

55.7 ± 7.0

a values in rows did not differ significantly p>0.05

Table 3. Whelping rate in the 2 treatment groups of bitches intravaginally or intrauterine inseminated with frozen-thawed semen

Treatment
groups

Group 1
intravaginal insemination
using infusion pipette

Group 2
intrauterine insemination
using endoscope

Whelping rate (%)

27.8a
(5/18)

68.7b
(11/16)

Different superscripts in the same row indicate significant difference p=0.039

Table 4. Litter size at birth and at weaning and percentage of male pups per litter in the 2 treatment groups of bitches. Mean ±SD (range)

Treatment
groups

Group 1
intravaginal insemination
using infusion pipette

Group 2
intrauterine insemination
using endoscope

Litter size at birth

3.0 ± 1.2a
(2-5)

4.9 ± 1.7b
(3-8)

Litter size at weaning

2.6 ± 0.9a
(2-4)

4.6 ± 1.7b
(2-8)

Percentage of male pups/litter (%)

51.5 ± 25.9a

54.7 ± 18.7a

Different superscripts in the same row indicate significant differences p<0.05

Figure 1. Distribution of litter sizes at birth in two treatment groups of bitches

Figure 2. Distribution of litter sizes at weaning in two treatment groups of bitches

DISCUSSION

Results of intravaginal insemination of bitches with frozen–thawed semen obtained by many authors were variable and usually unsatisfactory [4, 6, 7, 12, 16, 17]. Conception rates obtained in clinical or laboratory conditions ranged from 0% [4] to 50–60% [6, 15-17]. Therefore several methods of intrauterine insemination have been developed in bitches [1-3, 18, 21]. Norwegian method of AI includes the use of rigid catheters and transabdominal fixation of uterine cervix, which should be tilted in horizontal position [1]. This technique requires some training and is impractical in large, obese or nervous bitches. The risk of trauma and perforation of vaginal fornix should be taken into account. Recently used method of transcervical intrauterine AI uses the rigid endoscope for visualisation of vaginal portion of uterine cervix [21, 22]. The possibility of observation if the catheter has been properly introduced into cervical os is the obvious advantage of this technique. In the case of backflow of the semen catheter may be repositioned. Wilson [21] and Linde-Forsberg and al. [8] used this method of AI with success. However data concerning direct comparisons of this technique of AI with results of intravaginal insemination were scarce and equivocal [8, 15]. The present study showed that fertility of bitches after AI with frozen-thawed semen using endoscopic visualisation of vaginal portion of uterine cervix was significantly better when compared with results of intravaginal insemination. It should be noted that arbitrally chosen time of insemination at day 4 and 6 after LH peak i.e. 2 and 4 days after the onset of ovulation seems optimal for AI of bitches with cryopreserved semen. Oocytes are ovulated in bitches at stage of prophase of first meiotic division [5]. Consequently maturation of oocytes lasts for additional 48 hours after onset of ovulation. On the other hand, AI performed late in oestrus is advisable due to the short livability of frozen-thawed dog spermatozoa [9, 13]. Frozen-thawed sperm cells are in a state resembling capacitation and acrosome reaction (FAR-False Acrosome Reaction) which necessitates immediate fertilisation of ova [20]. Otherwise spermatozoa loose fertilisation ability and undergone cell death. Thus direct intrauterine deposition of frozen-thawed dog spermatozoa compensated partly for impaired metabolism and integrity of sperm cells deriving from cryopreservation procedures. Due the fertility of bitches inseminated intrauterine obtained in the present study is slightly lower when compared with results of AI with fresh dog semen, other methods of compensation for damage of cryopreserved sperm should be investigated. It seems that the main quantitative factor, which may improve results of AI of bitches with frozen-thawed semen is the increase of the number of spermatozoa deposited into the lumen of uterus.

Study was conducted within the realization scope of project KBN 3 P06K 004 23.

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Wojciech Niżański
Department and Clinic of Obstetrics,
Ruminant Diseases and Animal Health Care,
Wrocław University of Environmental and Life Sciences, Poland
pl. Grunwaldzki 49, 50-366 Wrocław, Poland
Phone: (+48 71) 32 05 315
email: nizanski@ozi.ar.wroc.pl

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