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			<p class="Type-of-Article" lang="en-GB">&nbsp;</p>
			<p class="Type-of-Article" lang="en-GB"><span class="CharOverride-1">Research Article</span></p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="title- ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="title- ParaOverride-1" lang="en-GB">Relationship of in vitro Cervical Mucus Penetration Assay with IgA and IgG Type Antibodies in Cross-bred Cows</p>
		</div>
		<div class="Basic-Text-Frame">
		  <p>&nbsp;</p>
		  <p class="Normal ParaOverride-2"><span class="CharOverride-3">Vikrant Jarora, Vinod Kumar Gandotra, Ranjna Sandhey Cheema, Amrit Kaur Bansal, Shahbaz Singh Dhindsa</span></p>
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		<div class="Basic-Text-Frame">
			<p class="Affiliations" lang="en-GB">Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India.</p>
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		<div>
			<p class="Abstract ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Abstract ParaOverride-1" lang="en-GB"><span class="CharOverride-5">Abstract</span> | IgG and IgA class antisperm antibodies were detected in cervical mucus of 25 crossbred cows with SperMar test. Sperm penetration through cervical mucus of these cows was also analysed <span class="CharOverride-6">in vitro</span>. Relationship of <span class="CharOverride-6">in vitro</span> CMPT with IgA and IgG type antibodies in cervical mucus was evaluated. Animals were grouped as G-1(&gt;40% IgG, IgA) and G-II (&lt; 40% IgG, IgA). IgG and IgA type antibodies were detected as 52.7±2.3%, 54.3±9.4% and 28.9±1.4%, 28.5±7.1 in cervical mucus of G-I and G-II, respectively. It indicated a significant (p&lt;0.05) difference of 23.8% and 25.8% in IgG and IgA class antibodies in cervical mucus among the two groups. A total of 54.9%, 56.8% of the tested cows had significant level of IgG and IgA in cervical mucus respectively. Sperm penetration of cattle bull spermatozoa in cervical mucus of 25 cows showed a variation. A mean distance covered by spermatozoa/ 30 min and number of spermatozoa penetrated in peak 0.5 cm in cervical mucus was 15.3±1.8 mm and 356.8±84.9, which ranged from 0-40 mm and 0-1414 respectively. Higher percentage of IgG, IgA did not have negative effect on <span class="CharOverride-6">in vitro</span> CMPT. Out of 25 cows, &gt; 40% IgA and IgG were detected in 13 and 17 cows respectively. Among the cows with higher percentage of IgA/IgG, there were only 6 cows (28%) that had higher level of both IgA and IgG (&gt;40%) and low values of CMPT. There were also one (3.8%) and four (15.3%) cows with only &gt; 40% IgA and IgG and low values of CMPT, respectively. It can be concluded that higher percentage of IgG-ASA and IgA-ASA reduced <span class="CharOverride-6">in vitro</span> penetration of spermatozoa through cervical mucus in 44% of the tested cross-bred cows. However, there was not any negative effect of ASA on <span class="CharOverride-6">in vitro</span> CMPT in a group of cows with &gt; 40% IgG/IgA in cervical mucus.</p>
		  <p class="Abstract ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Abstract ParaOverride-1" lang="en-GB"><span class="CharOverride-5">Keywords </span>| <span class="CharOverride-8">Cross-bred cows, Cervical mucus, IgG, IgA, CMPT</span></p>
		  <p class="Abstract ParaOverride-1" lang="en-GB">&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-9">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-5">Received</span> | October 15, 2014; <span class="CharOverride-5">Revised</span> | November 12, 2014; <span class="CharOverride-5">Accepted</span> | November 14, 2014; <span class="CharOverride-5">Published</span> | November 26, 2014&#9;&#9;</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-5">*Correspondence</span> | Ranjna Sandhey Cheema, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India; <span class="CharOverride-5">Email: </span>ranjna.cheema@gmail.com</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-5">Citation</span> | Jarora SV, Gandotra VK, Cheema RS, Bansal AK, Dhindsa SS(2014). Relationship of in vitro cervical mucus penetration assay with IgA and IgG type antibodies in cross-bred cows. Adv. Anim. Vet. Sci. 2 (11): 606-611.  </p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-9">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2014/2.11.606.611"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2014/2.11.606.611</span></a></p>
			<p class="Editor----Citation" lang="en-GB"><span class="Editor---Citation CharOverride-5" lang="en-US">ISSN (Online)</span> | 2307-8316; <span class="Editor---Citation CharOverride-5" lang="en-US">ISSN (Print)</span> | 2309-3331</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-9">Copyright </span>© 2014 Jarora et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Heading-1--Introduction----" lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" lang="en-GB">&nbsp;</p>
		  <p class="Heading-1--Introduction----" lang="en-GB">INTRODUCTION</p>
			<p class="Caps-on-First-Para ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1" lang="en-GB"><span class="_idGenDropcap-1">T</span>he cervical mucus (CM) functions as a guide for normal sperm to enter the uterus and may also act as a filter to prevent the passage of abnormal spermatozoa into the upper part of reproductive tract. Fertilizing capacity of spermatozoa has been shown to be strongly related to cervical mucus penetration test (CMPT) in bull<span class="CharOverride-13"> </span>(Tas et al., 2007) Impairment of cervical mucus penetration is an important component of the infertility effect of antisperm antibodies. Presence of IgA–antisperm antibodies (ASA), especially in cervical mucus is of great significance<span class="CharOverride-13"> </span>(Wang <span class="CharOverride-14">et al., 2009). ASA in seminal plasma or cervical mucus (CM)</span> impairs the ability of sperm cells to penetrate cervical mucus in human<span class="CharOverride-13"> </span>(Eggert-krust et al., 1991)<span class="CharOverride-13"> </span>and anti-fertility effect of ASA may be mainly due to immobilization of sperm in the CM (Check et al., 1994). Investigations in veterinary field<span class="CharOverride-15"> </span>regarding the influence of ASA in CM on fertility rates are very few. Therefore, relationship of <span class="CharOverride-6">in vitro</span> CMPT was analysed with IgA and IgG type antibodies, detected by SpermMar test in cervical mucus of cross-bred cows.</p>
			<p class="Caps-on-First-Para ParaOverride-1" lang="en-GB">&nbsp;</p>
		  <p class="Caps-on-First-Para ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" lang="en-GB">MATERIAL AND METHODS</p>
		  <p class="Heading-1--Introduction----" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">Procurement of Cervical Mucus</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Cervical mucus samples were collected<span class="CharOverride-5"> </span>from 25 cross-bred cows of dairy farm, Guru Angad Dev Veterinary and Animal Sciences University and private dairy farms around Ludhiana, Punjab, India.  At the time of estrus, CM was collected with the help of artificial insemination (AI) pipette in sterilized vials, mildly sonicated at 20 watts, inactivated at 56<span class="CharOverride-13">o </span>C for 30 min and stored in aliquots at -20<span class="CharOverride-13">o </span>C till further use. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">Procurement of Semen</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Fifty frozen semen straws of each of two bulls were procured from Semen Bank Bhattian, Khanna, Punjab, India.</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">Indirect Sperm Mar Test with Sperm Mar kit</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Indirect Sperm Mar test was performed as per the method of FertiPro<span class="CharOverride-13"> </span>(2011). Adherence of latex particles to different parts of spermatozoa in the presence of cervical mucus of cross-bred cow is shown in Fig 1. Diluted cervical mucus 1/4 with TALP medium, pH 7.4 and incubated at 37° C for 30 min, collected the motile sperms by centrifugation through histopaque, suspended the sperm pellet in TALP and adjusted the sperm concentration to 20 X 10<span class="CharOverride-13">6</span>. Incubated 100 µl of the sperm suspension of motile spermatozoa with 100 µl of inactivated 1/4 cervical mucus for 1hr at 37°C. Added 2 ml of TALP, mixed well and centrifuged for 10 min at 1500 rpm. Pellet was re-suspended with 50 µl of TALP. On a slide, mixed 10 µl of sperm suspension and 5 µl of Sperm Mar latex particles IgG / IgA, mixed, covered with cover slip, kept in humid chamber for 5 min and observed under bright field microscope at 400 X. Attachment of latex particles to the head / tail or whole sperm was observed. Adherence of latex particles to head, tail or both was noted and about 150 sperms in different fields were counted. Sum of all percent bound latex particle(s) from the various locations on the sperm surface per 100 spermatozoa was calculated:</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">%total bindino= (No. of sperms with bound lates particle/s)/(Totla no. of sperms counted) x 100</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB"><img class="_idGenPageitem-1" src="AAVS_MH20141015021045-Jarora-web-resources/image/Image23294392.png" alt="" /></p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-" lang="en-GB">Cervical mucus penetration test (Murase and Braun, 1990)</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">CMPT was performed as per the method of Murase and Braun<span class="CharOverride-13"> </span>(1990) in CM of 25 cows using pooled frozen-thawed semen.<span class="CharOverride-5"> </span>Cervical mucus was filled in a capillary by capillary action. Capillary was sealed from one side with polyvinyl alcohol powder and pre-heated at 37<span class="CharOverride-13">o</span> C for 10 min. Approximately 100 µl of frozen-thawed semen was placed at the bottom of an eppendorf tube and a capillary tube was placed with its open end in the semen. After 30 min of incubation at 37<span class="CharOverride-13">o </span>C, the capillary tube was fixed on scaled glass slide and viewed under bright field microscope at 400 X. The length of the tube was then scanned to establish the distance furthest from the semen reservoir attained by spermatozoa. The maximum distance of migration of spermatozoa after 30 min of incubation was defined as the migration distance. Number of migrated spermatozoa was counted in the peak 0.5 cm. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">Statistical analysis</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">In Sperm Mar test, 40% reaction between motile spermatozoa and coated latex particles of IgG and IgA is considered as lower limit of significant activity. Therefore animals were grouped as G-1(&gt;40% IgG, IgA) and G-II (&lt; 40% IgG, IgA). Relationship of IgA and IgG in cervical mucus with <span class="CharOverride-6">in vitro</span> CMPT was analyzed statistically according to Independent Sample T-Test.</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" lang="en-GB">RESULTS AND DISCUSSION</p>
		  <p class="Heading-1--Introduction----" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">Detection of IgG and IgA Type Antibodies by Sperm Mar Test</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Binding of latex particles to head, tail or both was detected in<span class="CharOverride-5"> </span>the presence of blood serum or cervical mucus, which indicated the development of ASA against sperm surface proteins (Figure 1). IgG and IgA type antibodies were detected as 52.7±2.3 %, 54.3±9.4% and 28.9±1.4 %, 28.5±7.1 in G-I and in G-II, respectively (Table 1). It indicated a significant (p&lt;0.05) difference of 23.8 % and 25.8% in IgG and IgA class antibodies in cervical mucus among the two groups. A total of 54.9 %, 56.8% of the tested cows had significant level of IgG and IgA in cervical mucus respectively.<span class="CharOverride-6"> </span>In general, the proportion of motile spermatozoa reacting in the SpermMar-IgA test is smaller than that reacting in the SpermMar-IgG test, but the contrary may occasionally occur<span class="CharOverride-13"> </span>(Ackerman et al., 1988), because IgA are of secretory type and less in quantity than IgG. In the present study, percentage of motile spermatozoa reacting in SpermMar-IgA and SpermMar-IgG test was almost same.</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">CMPT</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Distance covered by spermatozoa in cervical mucus/30 min and number of spermatozoa penetrated in peak 0.5 cm was evaluated in CMPT. Sperm penetration of cattle bull spermatozoa in cervical mucus of 25 cows showed a variation. A mean distance covered by spermatozoa/ 30 min and number of spermatozoa penetrated in peak 0.5 cm in cervical mucus was 15.3±1.8 mm and 356.8±84.9, which ranged from 0-40 mm and 0-1414 respectively.</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">Relationship of IgA/IgG and CMPT</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Since presence of ASA can inhibit passage of spermatozoa through cervical mucus, prevent membrane fluidity changes needed for capacitation, reduce the ability of spermatozoa to undergo the acrosome reaction, and interfere with binding to the zona pellucida and fertilization<span class="CharOverride-13"> </span>(Fijak and Meinhardt, 2006). Therefore, IgA and IgG in G-I and G-II were statistically compared with distance covered by spermatozoa in cervical mucus/30 min and number of spermatozoa penetrated in peak 0.5 cm in CMPT (Table 1). Distance covered by spermatozoa / 30 min was non-significantly (p&lt;0.05) higher in cows with &gt; 40% IgG, IgA (17.2±2.6, 19.2±2.6) than cows with &lt; 40% IgG, IgA (12.5±2.1, 11.1±1.9). Number of spermatozoa penetrated in peak 0.5 cm was also higher in cows with &gt;40% IGg, IgA (471.6±135.8, 408.7±124.4) than cows with &lt; 40% IgG, IgA (232.3±87.9, 278.9±104.5), respectively. It indicated that overall higher percentage of IgG, IgA did not have negative effect on <span class="CharOverride-6">in vitro </span>CMPT. Studies done in human also revealed variable effect of ASA on the acrosome reaction/ capacitation<span class="CharOverride-13"> </span>(Fijak and Meinhardt, 2006) and sperm binding to human zona pellucidae (Bronson et al., 1982; Bronson et al., 1990a; Bronson et al., 1990b). Presence of ASA can inhibit passage of spermatozoa through cervical mucus, prevent membrane fluidity changes needed for capacitation, reduce the ability of spermatozoa to undergo the acrosome reaction, and interfere with binding to the zona pellucida and fertilization (Fijak and Meinhardt, 2006). It can be predicted that although IgG and IgA were higher in animals but did not interfere with the cervical mucus penetration but may have interference with other processes i.e. capacitation, binding to zona pellucida or fertilization.<span class="CharOverride-15"> </span></p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" lang="en-GB">Correlation between <span class="CharOverride-2">in vitro</span> CMPT and Cervical Mucus-IgA and IgG</p>
			<p class="Body-Text" lang="en-GB">Although, higher percentage of IgG/IgA in G-I did not show any negative effect on CMPT, but a positive Pearson correlation of 0.26/0.16; 0.48/0.28 was obtained between distance covered by spermatozoa in 30 min/ spermatozoa penetrated in peak 0.5 cm and IgG;  IgA respectively. Out of 25 cows, &gt; 40% IgA and IgG were detected in 13 and 17 cows respectively. Among the cows with higher percentage of IgA/IgG, there were only 6 cows (28%) that had higher level of both IgA and IgG (&gt;40%) and low values of parameters of CMPT (Table 2). There were also one (3.8%) and four (15.3%) cows with only &gt; 40 % IgA and IgG and low values of CMPT, respectively. It can be interpreted from this observation that ASA developed in cervical mucus of cross bred cows were of immobilizing type in these cows. Antibodies in the cervical mucus bind to the antigens on the sperm surface and due to antigen-antibody reaction, head to head, head to tail or tail to tail agglutination occurs. Therefore, it indicated that IgA and IgG in cervical mucus may reduce the penetration of spermatozoa through cervical mucus by immobilizing or agglutinating the spermatozoa.<span class="CharOverride-17"> Study carried out by Wang </span>et al. (<span class="CharOverride-17">2009)</span><span class="CharOverride-18"> </span><span class="CharOverride-17">corroborated that p</span>resence of IgA-ASA, especially in cervical mucus is of great significance because the attachment of IgA antibodies to the sperm surface lowers sperm penetration ability, while IgG antibodies did not have the same effect<span class="CharOverride-17">. On the contrary our study revealed that IgA and IgG in cervical mucus are of equal importance in decreasing the penetration ability of spermatozoa.</span> There is strong evidence that in humans, sperm-mucus interactions can be affected by local ASA, especially of the IgA class, both under <span class="CharOverride-6">in vitro</span> and<span class="CharOverride-6"> in vivo </span>conditions<span class="CharOverride-13"> </span>(Eggert-Kruse et al., 1991). ASA in seminal plasma or CM impaired the ability of sperm cells to penetrate cervical mu<span class="Body-Text ParaOverride-1">cus (Eggert-Kruse et al., 1995). Check et al.<span class="CharOverride-13"> </span>(1994) demonstrated that the anti-fertility effect of ASA may be mainly due to immobilization of sperm in the CM. ASA have been shown to react with plasma membrane with variable biological effects<span class="CharOverride-13"> </span>(Chamley and Clarke, 2007).</span></p>
		  <p class="Body-Text" lang="en-GB">&nbsp;</p>
          
          <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150109202035.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150109202035.jpg" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/figures/20150109202035.jpg" target="new">Figure 1: </a></b><a href="http://nexusacademicpublishers.com/uploads/figures/20150109202035.jpg"><span>Showing binding of IgG/ IgA latex particles to spermatozoa in the presence of cervical mucus of a repeat breeding cow</a></p>
          </div>

			
			
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"><span class="CharOverride-5">Table 1: </span>Relationship of IgA and IgG class antibodies in cervical mucus with cervical mucus penetration test</p>
			<table width="657" height="315" class="Table-Style-1 TableOverride-1" id="table-1">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-2" />
					<col class="_idGenTableRowColumn-3" />
					<col class="_idGenTableRowColumn-4" />
					<col class="_idGenTableRowColumn-5" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-6">
						<td rowspan="2">
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-19">Type of Antibody</span></p>
						</td>
						<td rowspan="2">
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-19">Group Number</span></p>
						</td>
						<td rowspan="2">
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-19">Percentage (range)</span></p>
						</td>
						<td colspan="2">
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-19">CMPT</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-19">Distance (mm)/30 min</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-19">Sperm count in peak 0.5 cm</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td rowspan="2">
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">IgA (%)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">G-I</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">( n =15)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">45.69±2.95</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(30.7-57.6)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">19.2±2.6</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(2.0-28)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">471.6±135.8</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(0-1414)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">G-II</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(n=10)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">21.05±2.32</span><span class="Title CharOverride-22">b</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(17-37.1)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">11.1±1.9</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(0-23)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">232.3±87.9</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(0-1414)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td rowspan="2">
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">IgG (%)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">G-I</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(n =15)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">56.9±3.8</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(42.8-88.1)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">17.2±2.6</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(0-28)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">408.7±124.4</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(0-1414)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td height="59">
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">G-II</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">( n=10)</span></p>
						</td>
						<td>
						  <p class="Normal ParaOverride-1"><span class="Title CharOverride-21">31.75±1.63</span><span class="Title CharOverride-22">b</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(24.2-36.5)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">12.5±2.1</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(0-23)</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">278.9±104.5</span><span class="Title CharOverride-22">a</span></p>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">(0-1114)</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"><span class="CharOverride-12">Values with different superscripts are significant (p&lt;0.05)</span></p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
            
          <p class="Figure--and-Table-Heading" lang="en-GB"><span class="CharOverride-5">Table 2:  </span>Status of IgA and IgG type antibodies and CMPT in cross bred cows</p>
		  <table width="657" height="645" class="Table-Style-1 TableOverride-1" id="table-2">
				<colgroup>
					<col class="_idGenTableRowColumn-10" />
					<col class="_idGenTableRowColumn-11" />
					<col class="_idGenTableRowColumn-11" />
					<col class="_idGenTableRowColumn-11" />
					<col class="_idGenTableRowColumn-12" />
					<col class="_idGenTableRowColumn-13" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-6">
						<td rowspan="2">
							<p class="Normal"><span class="Title CharOverride-19">S. No.</span></p>
						</td>
						<td rowspan="2">
							<p class="Normal"><span class="Title CharOverride-19">Cow No.</span></p>
						</td>
						<td rowspan="2">
							<p class="Normal"><span class="Title CharOverride-19">IgA (%)</span></p>
						</td>
						<td rowspan="2">
							<p class="Normal"><span class="Title CharOverride-19">IgG (%)</span></p>
						</td>
						<td colspan="2">
							<p class="Normal"><span class="Title CharOverride-19">CMPT</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-14">
						<td>
							<p class="Normal"><span class="Title CharOverride-19">Distance covered by spermatozoa/30 min</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-19">Number of spermatozoa penetrated in peak 0.5 cm</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-15">
						<td>
							<p class="Normal"><span class="Title CharOverride-21">1.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1359**</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">43.1</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">439</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-23">11</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">77</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">2.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1214</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">57.6</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">88.1</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">16</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">380</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">3.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1383*</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">36</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">44.4</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">16</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">15</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">4.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1294**</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">57.6</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">88.1</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">11</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">110</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">5.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1358</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">20. 6</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">32.9</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">23</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">230</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">6.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1348</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">25.3</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">36.5</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">11</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">75</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">7.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1392*</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">18.6</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">54.4</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">10</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">274</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">8.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1391</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">30.4</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">32.3</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">10</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">89</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">9.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1396**</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">45.4</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">49.5</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">2</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">0</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">10.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1402**</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">47.8</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">50</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">17</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">230</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-17">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">11.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1355***</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">49.4</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">27.1</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">18</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">290</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">12.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1271</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">17</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">35</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">11</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">321</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">13.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1129</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">40.2</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">51</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">22</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">1291</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">14.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1386</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">69</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">63.2</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">40</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">1414</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">15.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1399</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">25</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">35.8</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">10</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">89</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">16.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1380**</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">54</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">53.9</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">15</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">115</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">17.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">KK7**</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">49.2</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">40.7</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">20</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">49</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">18.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">1959*</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">29.1</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">42.2</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">0</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">0</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-15">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">19.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">V- 155</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">40.5</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">61</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">28</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">1091</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">20.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">151</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">32.3</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">25.6</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">10</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">501</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">21.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">2199*</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">34</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">40</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">0</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">0</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">22.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">135</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">49.5</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">43.6</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">20</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">508</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">23.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">5206</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">37.1</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">28.7</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">10</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">80</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-18">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">24.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">8-JAS</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">56.8</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">66.1</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">30</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">577</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-15">
						<td>
							<p class="Normal"><span class="Title CharOverride-23">25.</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">44-JAS</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">32.5</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-23">24.2</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-21">22</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-21">1114</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"><span class="CharOverride-12">&gt;40% IgG and</span><span class="CharOverride-16"> </span><span class="CharOverride-12">&lt;25 mm distance covered by spermatozoa in 30 min/&lt;350 spermatozoa penetrated in peak 0.5 cm; **&gt; 40% IgA/ IgG and &lt;25 mm distance covered by spermatozoa in 30 min/&lt;350 spermatozoa penetrated in peak 0.5 cm and *** &gt;40% IgA and</span><span class="CharOverride-16"> </span><span class="CharOverride-12">&lt;25 mm distance covered by spermatozoa in 30 min/&lt;350 spermatozoa penetrated in peak 0.5 cm</span></p>
            
			<p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Therefore, it can be concluded that higher percentage of IgG-ASA and IgA-ASA reduced <span class="CharOverride-6">in vitro </span>penetration of spermatozoa through cervical mucus in 44% of the tested cross-bred cows, however, there was not any negative effect of ASA on <span class="CharOverride-6">in vitro</span> CMPT in a group of cows with &gt; 40% IgG/IgA in cervical mucus.</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" lang="en-GB">ACKNOWLEDGEMENTS</p>
		  <p class="Heading-1--Introduction----" lang="en-GB">&nbsp;</p>
			<p class="Body-Text" lang="en-GB">Authors are thankful to UGC, New Delhi for providing grant under major research projects. Authors are also thankful to semen freezing lab, GADVASU, Ludhiana, India for providing semen samples.</p>
			<p class="Body-Text" lang="en-GB">&nbsp;</p>
		  <p class="Body-Text" lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" lang="en-GB">REFERENCES</p>
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