EVALUATION OF THREE DIFFERENT SEMEN EXTENDERS FOR PRESERVATION OF DECCANI RAM SEMEN AT DIFFERENT INTERVALS
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Date
31-10-15
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PVNR TVU
Abstract
The aim of the present investigation wasto ascertain the efficacy of three
different semen extenders (Egg yolk citrate, Tris citrate fructose egg yolk extender and
Coconut milk extender) in preserving Deccani Ram Semen at varied intervals (0, 24 and
48 hours).The present investigation was undertaken by utilizing 10 Deccani rams aged
2-4 years stationed at ILFC, Rajendranagar, Hyderabad. Experiment was designed to
study the semen characteristics immediately after dilution (0 h) and also during storage
period at 24 (5ºC) and 48h (5ºC) by using conventional parameters (Colour, volume,
motility, concentration, livability, abnormalities, acrosome integrity, HOS-test, MBRT)
and assesed velocity of spermatozoa by using computer assisted sperm analysis (CASA)
and also to study the morphometryand morphological changes of ram spermatozoa
using imageJ analysis by electron microscopy (Scanning and Transmission electron
microscopy) with an objective of choosing suitable semen diluent which has better
sperm keeping quality
The overall mean scrotal circumference, semen ejaculate volume, sperm
concentration, mass activity, colour, consistency and mass activity of Deccani ram
semen were 25.90 cm, 0.63 ± 0.27 ml, 10825.00 ± 2742 millions per ml, creamy white,
thick creamy and 3.62 ± 0.05 (0-5 scale).In the present study, there was significant
correlation between scrotal circumference, semen volume and concentration.
A total of 144 ejaculates (18 ejaculates from each ram) were collected during the
study. Ram semen samples were distributed randomly to 3 groups - Group I (Egg yolk
citrate extender), II (Tris citrate fructose egg yolk extender) and III (Coconut milk
extender), at weekly interval for preservation.
The mean individual motility percentage of semen diluted in Group I, II and III
at 0 h were 70.93 ± 1.86, 75.10 ± 1.36 and 76.25 ± 1.05, at 24 h of storage (5ºC) were
58.43 ± 1.69, 63.22 ± 1.19 and 61.45 ± 0.84 and at 48 h of storage were 47.70 ± 1.27,
48.22 ± 1.70 and 46.45 ± 0.91, respectively. The mean live spermatozoa percentageof
semen diluted in Group I, II and III at 0 h were 77.00 ± 1.34, 79.50 ± 1.29 and 79.68 ±
0.99, at 24 h of storage (5ºC) were 66.66 ± 1.26, 67.95 ± 1.25 and 68.62 ± 1.27 and at
48 h of storage were 47.18 ± 1.19, 56.45 ± 0.90 and 50.87 ± 1.04, respectively.The
mean sperm abnormalities percentage of semen diluted in Group I, II and III at 0 h were
4.72 ± 0.23, 4.85 ± 0.16 and 4.47 ± 0.24, at 24 h of storage (5ºC) were 4.95 ± 0.19, 4.97
± 0.17 and 4.66 ± 0.18, respectively and at 48 h of storage were 5.41 ± 0.21, 4.93 ± 0.21
and 5.08 ± 0.23, respectively. The mean acrosome integrity percentageof Group I, II
and III after dilution (0 h) were 93.58 ± 0.58, 94.97 ± 0.54 and 93.52 ± 0.38, at 24 h of
storage (5ºC) were88.64 ± 0.74, 88.95 ± 0.80 and 87.02 ± 0.90 and at 48 h of storage
(5ºC) were 82.50 ± 5.51, 83.62 ± 5.54 and 79.10 ± 7.38, respectively.The mean
percentage of sperm positive to HOS test of semen diluted in Group I, II and III at 0 h
were 69.22 ± 1.88, 75.22 ± 1.32 and 71.85 ± 1.08, at 24 h of storage (5ºC) were60.20 ±
1.18, 60.60 ± 1.81 and 60.56 ± 1.00 and at 48 h of storage (5ºC) were 44.64 ± 0.92,
49.75 ± 0.98 and 44.45 ± 0.86, respectively.The mean MBRT time (min) ofsemen
diluted in Group I, II and III at 0 h were1.57 ± 0.06, 1.51 ± 0.06 and 1.51 ± 0.05, at 24 h
of storage (5ºC) were3.59 ± 0.07, 3.38 ± 0.08 and 3.74 ± 0.10 and at48 h of storage
(5ºC) were 6.17 ± 0.08, 6.34 ± 0.10 and 6.34 ± 0.10 respectively. The diluted semen (0
h) differed significantly (P≤0.05) between extenders pertaining to individual motility
and HOS test while at 24 h of storage, individual motility and MBRT differed
significantly (P≤0.05) between extenders and at 48 h of storage except sperm
abnormalities, MBRT and individual motility all the other conventional parameters
differed significantly (P≤0.05). Among them, Group II was significantly better.
The overall mean of all the parameters (excluding MBRT and sperm
abnormalities) for animals numbering from 1 to 8 were 71.82, 65.93, 70.91, 68.76,
69.07, 68.37, 68.58 and 65.44 respectively and based on which ranking was given to the
rams in the descending order of 1 > 3 > 5 > 4 > 7 > 6 > 2 > 8. The overall mean of all
the parameters (except sperm abnormalities and MBRT) for semen diluted in EYC,
TCFEY and CME were 67.01, 70.30 and 68.32 respectively. Based on which ranking
was given to the extenders in the descending order of TCFEY > CME > EYC.
Six experimental animals were selected and semen was collected, immediately
diluted and chilled to 5ºC. Computer assisted semen analysis (CASA; HTMIVOS v.
10.6; Hamilton–Thorne, Beverly MA, USA)was done at 1, 24 and 48 h of
storage.CASA parameters analysed were total motility, progressive motility, rapid
motility, medium motility, slow motility, static motility, elongation, sperm head area,
average path velocity (VAP), straightline velocity (VSL), curvilinear velocity (VCL),
straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head
displacement (ALH) and beat cross frequency (BCF).
The chilled (5ºC) semen at 1 h of storage differed significantly (P≤0.05)
between extenders pertaining to total motility, progressive motility, rapid motility and
BCF while at 24 h of storage only medium motility differed significantly (P≤0.05)
between extenders and at 48 h of storage VAP and VCL differed significantly
(P≤0.05).There was significant difference (P≤0.05) between the duration of storage (1,
24 and 48 h) for the parameters total motility, progressive motility, rapid motility,
medium motility, slow motility, static motility, VAP, VSL and VAP. Among them,
motility, progressive motility, rapid motility, medium motility, VAP, VSL and VAP
decreased significantly (P≤0.05) while slow and static motility increased significantly
(P≤0.05) as the period of storage increased from 1 to 48 h. There was no significant
(P≥0.05) difference between the duration of storage (1, 24 and 48 h) for parameters
sperm head area, elongation, STR, LIN, WOB, ALH and BCF.
The overall mean of all the CASA parameters (except wobble percentage) for
semen diluted in Group I, II and III were 57.92, 58.18 and 55.22 respectively. Based on
which ranking was given to the extenders in the descending order of TCFEY > EYC >
CME but there was no significant difference between EYC and TCFEY.The coefficient
of variation (CV) values for total motility, progressive motility, rapid motility, VAP,
VSL, VCL, STR, LIN, WOB, ALH, BCF, sperm head area and elongation were 12.50,
13.10, 14.62, 28.67, 31.74, 24.93, 19.33, 28.70, 24.77, 18.44, 18.76, 14.86 and 14.85
respectively. Based on CV of the CASA parameters, ram semen total motility (%) and
progressive motility (%) and sperm elongation (%) were more useful for assesment of
semen quality. The mean percentage of total motility of semen diluted in Group I, II and
III after chilling (5ºC) at 1 h were 82.38 ± 1.78, 89.52 ± 0.94 and 91.00 ± 0.68,
respectively. At 24 h were 80.38 ± 1.77, 78.08 ±1.82 and 79.69 ± 1.18, respectively. At
48 h were 77.77 ± 1.52, 79.44 ± 1.38 and 76.38 ± 1.98, respectively. The mean
percentage of progressive motility of semen diluted in Group I, II and III after chilling
(5ºC) at 1 h were 78.05 ± 2.08, 83.52 ± 1.10 and 86.01 ± 0.88, at 24 h were 76.97 ±
1.49, 74.91 ± 1.32 and 76.02 ± 1.24 and at 48 h were 73.61 ± 1.63, 73.61 ± 1.56 and
69.05 ± 1.67, respectively. The mean percentage for elongation semen diluted in Group
I, II and III after chilling (5ºC) at 1 h were 39.80 ± 1.15, 40.96 ± 0.90 and 40.07 ± 1.09,
at 24 h were 40.52 ± 1.09, 39.51 ± 0.94 and 40.72 ± 1.10 and at 48 h were 40.42 ±0.82,
40.45 ± 0.87 and 40.37 ± 1.01, respectively.
The morphometry of Deccaniram spermatozoa (n=12) obtained (two samples)
by electron microscopic studies using imageJ analysis were length (μm) of sperm head
was 7.87; width (μm) of sperm head was 4.32; Area (μm2) of sperm head was 26.85;
Perimeter (μm) of sperm head was 20.65; mid piece length (μm) was 14.03, proximal
mid piece width (μm) was 0.75; distal midpiece width (μm) was 0.51; volume of mid
piece (μm3) was 4.53 and acrosomal cap length (μm) was 5.24 ± 0.05 respectively.
When sperm morphological changes were observed by electron microscope after
dilution (0 h) with EYC, there were no much abnormalities of spermatozoa. Sperms
were normal with intact plasma membrane of the head and intact acrosome without
damage. At 24 h of storage at 5ºC, there was loss of intactness of the PM over the head
of spermatozoa, vesiculation of the outer acrosomal membrane (OAM) and plasma
membrane (PM). At 48 h of storage at 5ºC, most of the spermatozoa showed loss of the
acrosomal membrane and PM and damage to acrosomal cap. There was loss of
intactness of PM over the midpiece and the mitochondria showed large intracristal
spaces which marked the signs of degeneration.
Conventional parameters can be used for the routine semen evaluation for the
selection of best rams. If we afford to do semen analysis by CASA, the semen
evaluation accuracy can be increased. In this study semen quality was better upto 48 h
of storage, but some authors reported better fertility only upto 24 h of storage when
used for A.I. The findings of electron microscopic studies about sperm morphology of
the present study showed significant changes in the sperm morphology at 48 hrs but
there was no much significant difference between 1 and 24 h of storage. This might be
the reason for lower conception rates after 24 h of storage of liquid semen at 5ºC when
used for Artificial insemination.
Based on conventional parameters TCFEY showed better keeping quality but
based on CASA evaluation, EYC and TCFEY showed better keeping quality and there
was no significant difference between them compared to CME. So, based on this we
recommend TCFEY. But due to easy availability of ingredients and preparation, EYC
may be the best option for preservation of ram semen at 5ºC for the use of Artificial
insemination.
Description
Keywords
potassium, extraction, acidity, clay, sampling, planting, soil sampling, soil sciences, minerals, fractionation