**2. Assesment of in vivo fertility after AI of the cows**

**1.1. Collection of semen, dilution and processing**

64 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

x 106

Two extenders were used; Tris-egg yolk extender (T-EY)): 20 ml of chicken egg yolk and LDL extender: 8% LDL (w/v) in accordance with the method described by Moussa et al. (2002) (patent n° 0100292) [5]. The extenders were thawed on the day of sampling and main‐ tained at 37°C. Three bulls belonging to an artificial insemination centre and that had been approved for public use, were used. All three had a recorded progeny. Using the Laicophos® extender, the artificial insemination centre had a non-return rate at 60-90d of 64.7% for Bull 1, 57.2% for Bull 2, and 72.2% for Bull 3. The semen was collected using an artificial vagina. To excite the bulls, they were teased with a Normandy cow for thirty minutes prior to sam‐ pling. The semen was collected into a glass tube that had been previously warmed to 37°C. Following collection, the ejaculates were immediately placed in a water bath at 37°C. Each ejaculate was divided into two equal fractions. Each fraction was immediately diluted to 100

 spz/ml with the two extenders that had been previously warmed to 37°C, and then subjected to progressive cooling from 37°C to +4°C over 1h and 30 in a refrigerated unit be‐ fore being placed into straws. The semen was maintained in equilibrium for 4 hours at +4°C. The straws were held for 10 minutes at +4 cm from the surface of the liquid nitrogen

Before inseminating the cows, semen was evaluated on motility and plasma membrane in‐ tegrity. The semen was analysed using the Hamilton-Thorne sperm analyser with the CEROS 12 software program, Hamilton-Thorne biosciences, Inc, Beverly, USA. The machine had been previously configured for the analysis of bovine semen. The following parameters were studied: motility (% mobile spermatozoa), straight line velocity: VSL (μm/sec.), curvi‐ linear velocity: VCL (μm/sec.), the linearity index: LIN (= VSL/VCL x 100), amplitude of lat‐ eral head displacement: ALH (μm), and average path velocity: VAP (μm/sec). VAP, VSL, STR, and LIN provide information about the progressive movements of the spermatozoa, VCL and ALH characterise the lateral movements, and BCF (Beat Croix Frequency) pro‐

The post-thaw percentage of motile spermatozoa was greater in the LDL extender than in the Tris-egg yolk extender (table 1). The proportion of motile spermatozoa was nearly twice as high in the LDL extender, 58.3% vs. 46% in the Tris-egg yolk (table 1). To eval‐ uate plasma membrane integrity, semen was added to an hypo-osmotic solution (100 mOsm/kg H2O). The spermatozoa was observed under a phase-contrast microscope and classified as positive or negative. Positive spermatozoa (plasma membrane intact) tail swollen and / or curled:. Negative spermatozoa (plasma membrane damaged) tail not curled. No significant difference (table 2) was found between the semen that had been frozen-thawed in the LDL and Tris-egg yolk extenders. The results of the motility analy‐ sis and plasma membrane integrity demonstrated that the semen could be used by stock

(-120°C) before being immersed and then stored in liquid nitrogen (-196°C).

**1.2. Semen evaluation before artificial insemination in the field**

vides information about the frequency of movements.

breeders for artificial insemination.

One hundred and ninety-three females from 83 different herds were inseminated by three inseminators with 25 years of experience. The females included in the study were from dairy or suckler herds with a Calving to First Insemination Interval (CFI) of more than 60 days, heifers over 18 months old, and first inseminations only. For each insemination, the follow‐ ing data was recorded: date of insemination, herd number, the animal's identification num‐ ber, breed, lactation or calving index, date of the previous calving if relevant, condition score, the bull used, and the extender used. The pregnancy diagnoses were conducted by re‐ cording returns to oestrus and trans-rectal palpation between the 65th and 150th day of gesta‐ tion. This data is summarised in table 3. Pregnancies can be obtained in the field following the artificial insemination of cows with semen that has been frozen and thawed in the LDL extender. However, no significant difference could be found between the LDL extender and the Tris egg yolk extender in terms of the success rates of insemination (Table 4).


**Table 1.** Results of the motility of bovine spermatozoa following freezing and thawing in the LDL extender and in the Tris-egg yolk extender obtained using the Hamilton Thorne image analyser (n=3).The results given are the means ± standard deviation of the motility characteristics recorded for the three bulls.


success rate following insemination between the 2 extenders. The initial objective was not to demonstrate the superiority of the LDL extender, but to demonstrate its effica‐ cy in the field in terms of percentage gestation. The success rates with artificial insemi‐ nation are satisfactory (table 3): 59.2% for the LDL extender and 65.3% for the Trisegg yolk extender. In a previous study, Amirat et al.2004 [6] demonstrated that fertility was maintained *in vitro*. The hypoosmotic test was chosen to assess plasma mem‐ brane integrity as a proven correlation has been found between the results of the HOS test and the *in vivo* fertility rate [9]; the HOS test can therefore be used to predict fer‐ tility. Plasma membrane integrity was maintained with both the LDL and Tris-egg yolk extenders (table 2). These results concur with previous studies undertaken in the bo‐ vine species [10,11]. Around 60% of the spermatozoa that were frozen-thawed in the LDL extender presented with an alteration of the plasma membrane, whilst around 40% of the spermatozoa lost their motility. The percentage of spermatozoa with an al‐ tered plasma membrane may be higher than to the percentage or spermatozoa present‐ ing with a loss of motility. This implies that a certain number of spermatozoa may retain their motility with a damaged plasma membrane, this result agree with that re‐ ported by Salamon and Maxwell (1995) [11]. Nevertheless it is unlikely that such sper‐

Fertility Results After Artificial Insemination with Bull Semen Frozen with Low Density Lipoprotein Extender

http://dx.doi.org/10.5772/51868

67

Motility results demonstrate that the percentage of motile spermatozoa following thaw‐ ing is superior in the LDL extender in comparison with the Tris-egg yolk extender. These results concur with the works of Moussa et al. (2002) [5] and Amirat et al. (2004) [6]. However, inter-individual variability on the motility performances following thaw‐ ing has already been reported by Farrell et al. (1998) [12] and Holt (2000) [13]. The results obtained do not make it possible to relate the motility of the spermatozoa to fertility due to the insufficient number of measurements. No study has demonstrated a precise correlation between motility parameters and fertility in cows. In cattle, the percentage of mobile spermatozoa, linearity (LIN), and straight line velocity (VSL) seem to be correlated to fertility according to Budworth et al. (1988) [14], and Farrell et al. (1998)[12]. The average path velocity (VAP), curvilinear velocity (VCL), and the fre‐ quency of tail movements (FTM), also appear interesting [12]. According to Liu et al. (1991) [15], the most interesting motility parameters in human semen are linearity

(LIN), straight line velocity (VSL), and the percentage of rapid spermatozoa.

The confirmation of pregnancies were performed by rectal palpation on average at around the 100th day. However, embryonic mortality is recorded in the same way as failure of fertilisation; this reduces the fertility results observed. Ultrasonographic preg‐ nancy diagnosis at 30 days would have been more accurate for measuring the fertili‐ ty of the semen as the impact of embryonic mortality is lower between D0 and D30 than between D0 and D150. Descoteaux et al. (2006) [16] thus report that 10% of cows

**4. What parameters could influence the AI success rate?**

matozoa would be capable of crossing the zona pellucida.

**Table 2.** The effect of LDL and Tris-egg yolk extenders on the integrity of spermatozoal plasma membranes according to the HOS test N = sum of spermatozoa taken from Bulls 1, 2, and 3.


**Table 3.** Characteristics of the study population for each extender


**Table 4.** Effect of the extender used for freezing the semen on the success rate at insemination (as a %)
