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Effect of different levels and sources of zincsupplementation on quantitative and qualitative semen

attributes and serum testosterone level in crossbredcattle (Bos indicus × Bos taurus) bulls

Nishant Kumar, Ramesh Verma, Lallan Singh, Vijay Varshney, Ram Dass

To cite this version:Nishant Kumar, Ramesh Verma, Lallan Singh, Vijay Varshney, Ram Dass. Effect of different lev-els and sources of zinc supplementation on quantitative and qualitative semen attributes and serumtestosterone level in crossbred cattle (Bos indicus × Bos taurus) bulls. Reproduction Nutrition De-velopment, EDP Sciences, 2006, 46 (6), pp.663-675. <10.1051/rnd:2006041>. <hal-00900645>

Reprod. Nutr. Dev. 46 (2006) 663–675 663c© INRA, EDP Sciences, 2006DOI: 10.1051/rnd:2006041

Original article

Effect of different levels and sources of zincsupplementation on quantitative and qualitativesemen attributes and serum testosterone level incrossbred cattle (Bos indicus × Bos taurus) bulls

Nishant Ka, Ramesh Prashad Va, Lallan Prasad Sa,Vijay Prakash Vb, Ram Sharan Dc*

a Animal Reproduction Division, Indian Veterinary Research Institute, Izatnagar, 243 122, Indiab Division of Veterinary Physiology and Climatology, Izatnagar, 243 122, India

c Division of Animal nutrition, Izatnagar, 243 122, India

(Received 30 August 2005; accepted 7 July 2006)

Abstract – An experiment was conducted on 16 crossbred bulls (about 2 years of age, 316.2 ±0.77 kg average body weight), divided into groups I, II, III and IV to study the effect of differentlevels of Zn supplementation from inorganic and organic sources on semen quality. The animals inthe first 3 groups were supplemented with 0, 35 and 70 ppm Zn from Zn sulfate, respectively and theanimals in-group IV were supplemented with 35 ppm Zn as Zn propionate. Semen collection andevaluation was done in the first month (to assess semen quality at the start of the experiment) and7th, 8th and 9th month of experimental feeding to evaluate the effect of supplemental Zn on semenattributes. We gave 6 months for Zn feeding, so that 3 sperm cycles of spermatogenesis had passedand the collected semen reflected the complete effect of Zn supplementation. Six ejaculates fromeach bull were collected and evaluated for semen quantitative (ejaculate volume, sperm concen-tration and sperm number per ejaculate) and qualitative characteristics (semen pH, mass motility,individual motility, sperm livability percent and abnormal sperm percent, percent intact acrosome,bovine cervical mucus penetration test, hypo-osmotic sperm swelling test) and activity of seminalplasma enzymes i.e., alkaline phosphatase, acid phosphatase, GOT and GPT. Testosterone levelin the blood serum of crossbred bulls was also estimated. Mean values of semen quantitative andqualitative characteristics at the start of the experiment were statistically non significant (P > 0.05)in all the crossbred cattle bulls, however, there were statistically significant differences among thebulls of different groups after 6 months of zinc supplementation. Mean ejaculate volume (mL)was 2.37, 4.70, 5.86 and 6.38, respectively in groups I to IV, indicating a statistically significant(P < 0.05) higher semen volume in Zn-supplemented groups as compared to the control group ofbulls. Similarly, sperm concentration (million.mL−1), live sperm (%) and motility (%) were signifi-cantly (P < 0.01) higher in Zn-supplemented groups as compared to the control group. The resultsof BCMPT and HOSST revealed a significant improvement in sperm functional ability in all thegroups supplemented with Zn as compared to the control group. The activity of alkaline and acidphosphatase in seminal plasma was significantly (P < 0.05) higher in the Zn-supplemented groups,whereas GOT and GPT activities in seminal plasma were significantly (P < 0.05) lower in theZn propionate supplemented group as compared to the control group. Testosterone concentration(ng.mL−1) in blood serum was significantly higher in animals of groups III and IV, as comparedto control group. It may be concluded that Zn supplementation either in the inorganic or organicform in the diet of crossbred bulls improved the qualitative and quantitative attributes of semen;

* Corresponding author: rsd@ivri.up.nic.in

Article published by EDP Sciences and available at http://www.edpsciences.org/rnd or http://dx.doi.org/10.1051/rnd:2006041

664 N. Kumar et al.

however, the number of sperm per ejaculate, mass motility and semen fertility test like bovine cervi-cal mucus penetration was significantly higher in bulls given Zn in an organic form (Zn propionate)as compared to an inorganic form (Zn sulfate).

zinc / crossbred bulls / semen / sperm / testosterone / seminal plasma enzyme

1. INTRODUCTION

Zinc is an essential trace element re-quired for the action of more than 200 met-allo enzymes and plays an important role inpolymeric organization of macromoleculeslike DNA and RNA, protein synthesis andcell division [1]. Zinc plays an importantrole in prostate, epididymal and testicu-lar functions [2]. Zinc has been reportedto influence the process of spermatogen-esis [3], controls sperm motility [4], sta-bilizes sperm membrane [5], preservesthe ability of sperm nuclear chromatinto undergo decondensation and modulatessperm functions [6]. Hypozinkemia leadsto gonad dysfunction, decreased testicu-lar weight, atrophy of seminiferous tubulesand complete cessation of spermatogene-sis [7]. Zinc is found in high concentrationin the male reproductive tract as well as insemen [8]. The mean concentration of Znin the semen of bulls, rams, stallions andboar has been reported as 83.15 ± 61.61,60.46± 35.37, 86.20± 45.88 and 171.74±65.72 mg.kg−1 wet weight of tissue, re-spectively [9]. The recommended level ofzinc in the diet of cattle is 35–40 ppmand is sufficient for normal body functions,but for enhanced immunity, higher levelsof zinc have been found beneficial [10].Similarly, supplementation of zinc as or-ganic/chelated minerals has been foundmore beneficial as compared to inorganicsources [11–13]. Till date, very little workhas been done in crossbred cattle bulls inrelation with zinc supplementation. More-over, the effect of organic zinc supplemen-tation on semen quality has so far not beenstudied. Most of the studies on zinc supple-mentation and its effect on semen qualityhave been conducted in men [8, 14, 15]

and very little information is available re-garding zinc supplementation and spermfunction test in cattle.

Therefore, the present experiment wasconducted to study the effect of higherlevels of Zn supplementation from zincsulfate and zinc propionate on the quan-titative and qualitative characteristics ofsemen along with testosterone level in theblood serum of crossbred cattle bulls.

2. MATERIALS AND METHODS

2.1. Animals, their feedingand management

This study was conducted on sixteenyoung and healthy crossbred cattle (Bos in-dicus × Bos taurus) bulls of about 2 yearsof age, having 316 ± 0.77 kg mean bodyweight, divided randomly into groups I,II, III and IV, comprised of four animalsin each group. All the bulls were main-tained in the Animal Nutrition Division ofIndian Veterinary Research Institute, Izat-nagar, and fed on wheat straw and concen-trate mixture in the ratio of 1:1 to meettheir dry matter and crude protein require-ment [10]. The basal diet (wheat strawand concentrate mixture) had 32.54 ppmzinc. Concentrate mixture was comprisedof crushed maize grain (30%), soybeanmeal (25%), wheat bran (42%), mineralmixture (2%) and common salt (1%). Inaddition, the animals were given 0, 35 and70 ppm zinc as zinc sulfate in the first3 groups, respectively, whereas, 35 ppmzinc as zinc propionate was given to bullsin group IV. All the bulls were kept ina well ventilated shed having a cemented

Effect of zinc supplementation on semen quality 665

floor and arrangements for individual feed-ing. Clean and fresh drinking water wasprovided twice daily i.e. at 10.00 am and3.00 pm to all the animals. Body weights ofall the animals were recorded at an intervalof 15 days for the formulation of diet andcalculation of zinc to be supplemented inthe diet of each animal. This feeding prac-tice lasted for 9 months.

2.2. Collection of semen and evaluationof its quantitative and qualitativecharacteristics

The semen from all the groups of bullswas collected in an artificial vagina overa dummy or male partner in the first onemonth to assess semen characteristics ofall bulls at the start of the experiment. Inthe last 3 months of experimental feeding,6 ejaculates from each animal were col-lected to study the effect of Zn supplemen-tation on semen characteristics. We waitedfor 6 months to assess the effect of zincsupplementation on semen attributes sincethe time required to complete one cycle ofspermatogenesis (for complete sperm for-mation and maturation) is 60 days in bulls,so around 3 sperm cycles were passedwith zinc supplementation in the diet. Thiswas done in order to ensure that the col-lected semen reflected complete effect ofZn supplementation. All semen sampleswere evaluated for quantitative and quali-tative characteristics along with separationof seminal plasma, by centrifuging the se-men at 3000 rpm for 20 min, for estimationof seminal plasma enzyme activity.

2.2.1. Evaluation of quantitativecharacteristics of semen

Quantitative characteristics of semen in-cluded ejaculate volume, sperm concen-tration, and sperm number per ejaculate.Ejaculate volume (mL) of semen was

recorded to the nearest 0.1 mL in a gradu-ated glass tube. The concentration of sperm(million.mL−1) in the fresh semen was de-termined using a haemocytometer [16].The number of sperm present in 80 smallsquares was counted and the total num-ber was multiplied by 10 millions to getthe sperm concentration per mL of semen.Sperm number per ejaculate was calculatedsimply by multiplying the concentration ofsperm by total volume of semen.

2.2.2. Evaluation of qualitativecharacteristics of semen

It included the assessment of semenpH, mass motility, individual motility,sperm livability percentage and abnormalsperm percentage, intact acrosome per-centage, two sperm function tests viz.,bovine cervical mucus penetration test(BCMPT) and the hypo-osmotic spermswelling test (HOSST), and estimation ofseminal plasma enzyme activity i.e. alka-line phosphatase, acid phosphatase, glu-tamic oxaloacetic transaminase [GOT] andglutamic pyruvic transminase [GPT]. Se-men pH was noted immediately after col-lecting the semen using a digital pH meter(Century, India). Mass motility of semenwas graded from a 0–5 scale, based onthe appearance of waves and swirls cre-ated by sperm movement when visualizedby keeping one drop of semen on a glassslide, without cover slip, under low powermicroscopic magnification (10×) [16]. Ex-tremely rapid waves or swirl motion ofsperms were given a 5 numerical scale,comparatively slower waves and swirlswere given a 4 numerical scale and like-wise slow moving, extremely slow mov-ing, no movement and non-motile spermwere given 3, 2, 1 and 0 numerical scalesrespectively. The individual motility offreshly diluted semen was assessed aftercovering a semen drop on a glass slidewith a thin cover slip at 37 ◦C, under high

666 N. Kumar et al.

power magnification (40×). The individualmotility was recorded as the percentage ofprogressive motile sperm. Sperm livabilitypercentage was calculated by using Eosin-Nigrosin stain. Similarly, the abnormalsperm percentage was calculated by usingRose Bengal stain under high power mag-nification (100×) [17]. Percent intact acro-some was assessed by staining the semensmears with Giemsa stain [18]. The bovinecervical mucus penetration test (BCMPT)was carried out by following the proceduredescribed earlier [19], in which distance(mm) traveled by progressive sperm wasmeasured under high power microscopicmagnification (40×), by allowing sperm totravel in a capillary tube, filled with cer-vical mucus of a cyclic cow, at 37 ◦C for60 min. The percentage of a hypo-osmoticswollen sperm was observed by incubat-ing semen with a hypo-osmotic solutionat 37 ◦C for 60 min and examining theswelling of the sperm tail under high powermicroscopic magnification (40×) for thehypo-osmotic sperm swelling test [20].

Alkaline phosphatase [21], acidphosphatase [22], glutamic oxaloacetictransaminase [GOT] and glutamic pyruvictransminase [GPT] activity [23] in seminalplasma was estimated using diagnostickits (Glaxo), manufactured by SigmaDiagnostic Pvt. Ltd., Baroda, India.

2.3. Collection of blood and estimationof serum testosterone level

Blood samples were collected from allfour groups of bulls at monthly intervalsand serum was separated by centrifuga-tion of samples at 3000 rpm for 20 min.The separated serum was stored at –20 ◦Cin sterilized glass vials for estimation oftestosterone. Testosterone concentration inblood serum samples was determined us-ing RIA kits supplied by Immunotech,France. The unknown samples and stan-dard samples were incubated with 125I

serum-labeled testosterone in antibody-coated tubes. After incubation, the liquidcontent of the tubes was aspirated and thebound radioactivity was determined in agamma counter (Packard, USA). A stan-dard curve was prepared with 6 standardsand testosterone concentration in unknownsamples was obtained from the curve by in-terpolation.

2.4. Statistical analysis

The data collected during the period ofstudy were analyzed as per method de-scribed by Snedecor and Cochran [24],using one-way analysis of variance andsignificant means were compared using theDuncan multiple range test [25].

3. RESULTS

3.1. Semen characteristics of bulls at thestart of the experiment

The mean values of quantitative (ejacu-late volume (mL), concentration of sperm(million.mL−1) and number of spermper ejaculate (million)) and qualitativecharacteristics (semen pH, mass motil-ity (0–5 points scale), individual motil-ity (%), sperm livability (%), abnormalsperm (%,) intact acrosome (%), BCMPT(mm), HOSST (%), GOT (unit.mL−1),GPT (unit.mL−1), alkaline phosphatase(KAU.100 mL−1) and acid phosphatase(KAU.100 mL−1)) of semen of 16 cross-bred cattle bulls at the start of experimentare presented in Table I and the results re-vealed no significant difference in any ofthese parameters.

3.2. Semen characteristics of bulls after6 months of zinc supplementation

The mean values of semen quanti-tative and qualitative characteristics and

Effect of zinc supplementation on semen quality 667

Table I. Semen quantitative and qualitative characteristics of crossbred bulls at the start of theexperiment.

Quantitative Characteristics Mean (± SE)

Semen volume (mL) 2.84 ± 0.13

Sperm concentration (million.mL−1) 785 ± 62.1

Sperm number per ejaculate (millions) 2229 ± 226.9

Qualitative characteristics Mean (± SE)

Semen pH 6.64 ± 0.04

Mass motility (0–5 scale) 3.15 ± 0.96

Individual motility (%) 75.11 ± 1.35

Sperm Livability (%) 77.05 ± 1.24

Abnormal sperm (%) 12.0 ± 1.10

Intact acrosome (%) 78.69 ± 1.35

BCMPT (mm)* 15.94 ± 2.05

HOSST (%)** 48.44 ± 2.53

Alkaline phosphatase (KAU***.100 mL−1) 135.21 ± 12.73

Acid phosphatase (KAU.100 mL−1) 254.89 ± 26.87

Glutamic oxaloacetic transaminase (unit.mL−1) 492.38 ± 2.82

Glutamic pyruvic transaminase (unit.mL−1) 35.24 ± 0.72

* mm: Mean penetration distance traveled by bull sperm in cyclic bovine cervical mucus.** % of sperm reactive to hypo-osmotic sperm swelling test.*** KAU: King and Armstrong Unit. It is defined as the quantity of phosphatase that acting upon dis-odium phenyl phosphate in excess at pH 9 for 30 min liberates 1 mg of phenol. It is the standard ofmeasure devised by King and Armstrong. For Alkaline and acid phosphatase 1 KAU.dl−1 = 1 IU/L/7.Therefore, 7 KAU.dl−1 = 1 IU.L−1.

blood serum testosterone concentration(ng.mL−1) in crossbred cattle bulls of dif-ferent groups after 6 months of Zn supple-mentation are presented in Table II.

3.2.1. Quantitative characteristicsof semen

The results revealed that mean ejac-ulate volume (mL), sperm concentration(million.mL−1) and sperm number perejaculate (millions) in different groups ofcrossbred cattle bulls were affected pos-itively due to Zn supplementation. Meanejaculate volume (mL) was significantly(P < 0.05) lower in-group I (control) ascompared to all Zn-supplemented groups

(groups II, III and IV) and increased sig-nificantly with an increase in level of Znin the diet; however, there was no signif-icant difference in-group III (70 ppm Znas Zn sulphate) and group IV (35 ppmZn as Zn propionate). Sperm concentra-tion (million.mL−1) and sperm numberper ejaculate (million) were significantly(P < 0.01) higher in the Zn-supplementedgroups as compared to the control. Thoughthe sperm concentration (per mL) was sta-tistically alike in groups III and IV, thesperm number per ejaculate was signifi-cantly (P < 0.01) higher in group IVas compared to the 3 other groups, whichindicated a better effect of organic Zn(Zn propionate) as compared to inorganicZn (Zn sulphate) on sperm production.

668 N. Kumar et al.

Tabl

eII

.Sem

enqu

antit

ativ

ean

dqu

alita

tive

char

acte

rist

ics

and

bloo

dse

rum

test

oste

rone

leve

lin

cros

sbre

dbu

llsaf

ter6

mon

ths

ofzi

ncsu

pple

men

tatio

n.

Sem

ench

arac

teri

stic

s

Qua

ntit

ativ

eG

roup

IG

roup

IIG

roup

III

Gro

upIV

Eja

cula

tevo

lum

e(m

L)*

2.37±0

.044

a4.

70±0

.112

b5.

86±0

.114

c6.

38±0

.182

c

Sper

mco

ncen

trat

ion*

*(m

illio

n.m

L−1

)76

0.83±1

4.09

a10

12.0

8±8

.702

b14

09.5

8±1

7.90

1c14

72.0

9±1

7.63

1c

Sper

mnu

mbe

rpe

rej

acul

ate

(mil

lion/e

jacu

late

)**

1806

.97±3

8.63

a47

56.7

7±1

43.7

4b82

46.0

43±1

19.0

3c93

91.8

7±1

12.5

4d

Qua

lita

tive

pH**

6.75±0

.013

a6.

73±0

.010

a6.

63±0

.013

b6.

61±0

.015

b

Mas

sm

otil

ity

(0-5

)**

2.71±0

.112

a3.

37±0

.101

b3.

96±0

.042

c4.

33±0

.098

d

Indi

vidu

alm

otil

ity

(%)*

72.5

8±0

.462

a77

.25±0

.819

b83

.37±0

.756

c88

.04±0

.641

c

Liv

esp

erm

(%)*

73.4

6±0

.609

a80

.65±0

.742

b86

.62±0

.387

c87

.31±0

.758

c

Abn

orm

alsp

erm

(%)

13.1

7±0

.322

12.2

9±0

.351

11.9

4±0

.432

12.5

3±0

.210

Inta

ctac

roso

me

perc

enta

ge**

76.0

6±0

.48a

81.1

7±0

.61b

86.5

0±0

.50c

87.0

4±0

77c

Bov

ine

cerv

ical

muc

uspe

netr

atio

nte

st(B

CM

PT)

(mm

)**

11.5

2±0

.25a

16.4

3±0

.34b

24.7

9±0

.40c

29.6

0±0

.420

d

Hyp

oos

mot

icsp

erm

swel

ling

test

**(H

OSS

T)

46.2

1±0

.44a

53.4

0±0

.32b

66.3

8±1

.16c

67.6

0±1

.11c

Alk

alin

eph

osph

atas

eac

tivit

y**

(KA

U.1

00m

L−1

)13

1.12±2

.54a

140.

0±0

.59b

258.

46±1

.59c

262.

50±1

.45c

Aci

dph

osph

atas

eac

tivit

y**

(KA

U.1

00m

L−1

)21

8.13±2

.32a

260.

87±1

.13b

341.

58±2

.66c

349.

16±2

.47c

Glu

tam

icox

aloa

ceti

ctr

ansa

min

ase

(GO

T–

unit

.mL−1

)*49

9.42±3

.49a

495.

29±3

.66b

482.

29±1

.54b

470.

04±2

.21b

Glu

tam

icpy

ruvi

ctr

ansa

min

ase

(GPT

–un

it.m

L−1

)*36

.29±0

.66a

33.3

3±0

.61b

30.0

0±0

.77c

29.2

5±0

.44c

Seru

mte

stos

tero

ne(n

g.m

L−1

)*2.

18±0

.46a

2.38±0

.33a

3.17±0

.50b

3.52±0

.58b

*P<

0.05

;**

P<

0.01

.a,b,

c,d

Mea

nsw

ith

diff

eren

tsup

ersc

ript

ina

row

diff

ersi

gnifi

cant

ly.

Effect of zinc supplementation on semen quality 669

3.2.2. Qualitative characteristicsof semen

The result revealed significantly (P <0.01) lower semen pH in-group III andgroup IV as compared to groups II andI, but values were with in normal range.The mean mass motility values (0–5 scale)were significantly (P < 0.01) differentbetween all four groups. Mean individualsperm motility (%) in different groups ofcrossbred bulls differed significantly (P <0.01), indicating significantly (P < 0.01)higher sperm motility in Zn-supplementedgroups. The highest mean livability (%)was recorded in-group IV, which was sig-nificantly (P < 0.01) higher than groups IIand I and non-significantly (P > 0.05)higher than group III. The results revealedno significant difference in mean abnor-mal sperm (%) in four groups of cattlebulls. The results revealed a progressive in-crease in intact acrosome (%), indicatinga significantly (P < 0.01) higher intactacrosome percentage in Zn-supplementedgroups as compared to the control group.The BCMPT indicated that mean penetra-tion distance (mm) traveled by the cross-bred bull sperm in the bovine estrus mu-cus was maximum in group IV (29.60)followed by group III (24.79), group II(16.43) and group I (11.52). The differencein penetration distance among all groupswas significantly (P < 0.01) different.The HOSST indicated higher mean per-centage of hypo osmotic swollen spermsin-group IV (67.60), followed by group III(66.38), group II (53.40) and group I(46.21). The values of HOSST in Zn-supplemented groups differed significantly(P < 0.01) from the control group, but val-ues between group III and group IV werestatistically alike. The mean activities ofalkaline phosphatase and acid phosphatase(KAU.100 mL−1) in seminal plasma weresignificantly (P < 0.01) higher in Zn-supplemented groups as compared to thecontrol group. The results further revealed

that there was a progressive significant(P < 0.01) decrease in GOT and GPT ac-tivity (unit.mL−1) in crossbred bulls fromgroup I to group IV, indicating a signifi-cant difference in the Zn-supplemented andcontrol group. Mean serum testosteroneconcentration (ng.mL−1) were found to be2.18, 2.38, 3.17 and 3.52 in 4 groupsof crossbred cattle bulls, respectively. Nosignificant difference was observed ingroups I and II but significantly (P < 0.05)higher values were found in-group III andgroup IV as compared to the control group.

4. DISCUSSION

In this study, the effect of different lev-els and sources of zinc supplementation onsemen quantitative and qualitative charac-teristics and blood serum testosterone levelof crossbred cattle bulls was studied. Theresults revealed that supplementation of Znin the inorganic form (Zn sulphate) and or-ganic form (Zn propionate) improved thesemen quality of bulls as compared tothe non-supplemented control group. Znpropionate was better than Zn sulfate inalmost every character of semen studied.This might be due to the fact that Zn propi-onate has got more bioavailability than Znsulfate, as a result, there might have beenmore absorption, distribution and uptakeof Zn in the Zn propionate supplementedgroup, which accounts for its better effectover Zn sulfate. Our study is perhaps thefirst study correlating Zn propionate andsemen quality of crossbred cattle bulls.

4.1. Semen quantitative and qualitativecharacteristics of crossbred cattlebulls at the start of the experiment

At the start of the experiment, semenwas collected from all 16 bulls and eval-uated for quantitative and qualitative char-acteristics. The results revealed that all the

670 N. Kumar et al.

bulls were having normal semen quality.The mean values of quantitative and qual-itative seminal parameters did not differsignificantly among bulls at the start of theexperiment.

4.2. Semen quantitative characteristicsof crossbred cattle bulls after6 months of Zn supplementation

The results revealed significantly (P <0.05) higher values of ejaculate volume ingroups supplemented with Zn sulfate andZn propionate as compared to the con-trol group of bulls. The present resultsare in agreement with the findings of ear-lier researchers who observed increasedsemen volume when they supplemented Znsulfate in the diet of goats [26] and rab-bits [27, 28]. However, no published reportwas available in the literature on the ef-fect of Zn propionate supplementation onsemen quality in ruminants; probably thisis the first report showing the effect ofzinc propionate supplementation on semenof crossbred cattle bulls. Semen volumemainly constitutes secretion of the testes,epididymis and accessory sex glands, espe-cially prostate gland. Zn has been reportedto stimulate growth and development ofprimary, secondary and accessory sex or-gans as evidenced by atrophy of theseorgans in rams, when they were fed a Zndeficient diet [7]. The main source of zincin the semen is the prostate gland wherethe highest concentration of Zn has beendemonstrated, and it acts as a marker ofprostatic functions [29, 30]. So, enhancedsemen volume by Zn supplementation maybe attributed to increased secretory activ-ity of prostatic cells, since 35–40% se-men volume is contributed by the prostategland. In the present study, we recordeda highly significant (P < 0.01) increasein sperm concentration and sperm numberper ejaculate in all the Zn-supplementedgroups as compared to the control group,

which indicated a beneficial effect of Znon spermatogenesis. Similar results wereobserved in men [3], rams [5], bucks [26]and rabbits [27], when Zn was supple-mented in their diet. This may be due tothe fact that Zn plays an indispensablerole in spermatogenesis. The production ofsperm necessitates extensive cell divisionand Zn plays a significant role in it byinfluencing mitotic and meiotic cell divi-sions, along with synthesis of DNA andRNA by enhancing the activity of DNApolymerase and RNA polymerase, the twoZn containing enzymes. Zn also helps inencoding a transcription factor involvedin spermatogenesis [31]. Zn is also in-volved in the activation and maintenanceof the germinal epithelium of semineferoustubules and also stimulates production andsecretion of testosterone, which influencesspermatogenesis [3]. Moreover, most im-portant enzymes involved in the processof spermatogenesis are sorbitol dehydro-genase and lactate dehydrogenase, whichare essentially zinc metalloenzymes [32].All these factors may account for improvedsperm concentration and sperm number perejaculate in Zn-supplemented groups.

4.3. Semen qualitative characteristicsof crossbred cattle bulls after6 months of zinc supplementation

The results revealed significantly (P <0.01) lower semen pH in groups III and IV,as compared to groups I and II, but valueswere with in the normal pH range. Theseresults are contradictory with those of [26],who reported an increased pH value in Zn-supplemented bucks. The difference in pHvalues may be due to the species differenceand also the short duration of zinc sup-plementation in their study as comparedto long term Zn supplementation in thepresent study. In our study, reduced semenpH might be attributed to increased acidicsecretion of the prostate gland in semen 39.

Effect of zinc supplementation on semen quality 671

Supplementation of Zn in the diet of cattlebulls revealed significant improvement inboth mass and individual motility in all Zn-supplemented groups as compared to thecontrol group. Our results were in agree-ment with previous reports in men [3],sheep [5] and rabbits [27]. Improved spermmotility may be due to the fact that theprimary donor of energy needed by thesperm flagella for movement is ATP andZn controls the motility of sperm by con-trolling energy utilization through the ATPsystem, through regulation of phospho-lipid energy reserves and improving spermoxygen uptake [33]. Another reason forthe enhancement of sperm motility in Zn-supplemented groups may be the increasedactivity of Zn containing enzymes viz.sorbitol dehydrogenase and lactate dehy-drogenase which play significant roles insperm motility [30]. Zn is also a scavengerof free oxygen radical and protects spermfrom oxidative damage and lipid per ox-idation by inhibiting phospholipase [32].Thus, the anti-oxidant action of Zn maybe responsible for improved motility ofsperm in Zn-supplemented groups. In thepresent study, it was observed that supple-mentation of Zn above the recommendedlevel in the diet of crossbred cattle bulls re-sulted in a highly significant (P < 0.01)increase in live sperm percentage. Our re-sults were in accordance with the findingsof earlier researchers in the rabbit [33]and man [34], who reported improved livesperm percentage by Zn supplementation.Improved livability of sperm may be dueto the membrane stabilizing action of Zn,by virtue of which, it prevents leakage ofenzymes, proteins and other vital compo-nents of sperm, thus extending the func-tional life of sperm. Moreover, Zn alsostabilizes ribosomes, lysosomes, DNA andRNA, which help in survival and normalfunctioning of the sperm [35]. They furtherreported that Zn protects sperm from freeradical induced damages by scavenging ex-cessive free radicals and thus improving

sperm viability. Bires et al. [36] reportedthat Zn, as a constituent of a large numberof metalloenzymes, is involved in severalenzymatic reactions associated with car-bohydrate, protein, lipid and nucleic acidmetabolism, which may account for im-proved sperm livability. Moreover, Zn hasbeen reported as a primary factor responsi-ble for the production of an anti-bacterialsubstance released from the prostate glandinto semen [39], which may also accountfor improved live sperm percentage. Ab-normal sperm (%) present in the semenof different groups of bulls varied from11–13%, indicating that Zn supplemen-tation did not affect this character. Thepresent findings were in agreement withthose of [37, 38], who also did not observeany change in abnormal sperm percentage,when they supplemented Zn in the dietof men and boars, respectively. The nor-mal and abnormal morphology of sperm iscompletely dependent on the spermiogene-sis phase of spermatogenesis, which in turnis regulated by Sertoli cells [39] and Zndeficiency did not have any structural andfunctional changes on sertoli cells [40].Since there was no effect of zinc on Ser-toli cells, so, no significant difference wasobserved in abnormal sperm percentage indifferent groups of crossbred cattle bulls.

Intact acrosome percentage was sig-nificantly (P < 0.01) higher in Zn-supplemented groups as compared to thecontrol group. Improved intact acrosome(%) in Zn-supplemented groups may beattributed to anti-oxidant properties andmembrane stabilizing action of Zn modu-lates the stability of the acrosomal mem-brane by inhibiting lipid per oxidation byinfluencing phospholipase, resulting in afluidity change [32]. Zn has been foundto stabilize various acrosomal enzymeslike acrosin, acid phosphatase and phos-pholipase, which may account for im-proved intact acrosome percentage. More-over, acrosome is a highly specialized formof lysosomes and Zn has been reported

672 N. Kumar et al.

to stabilize and inhibit libilization of lyso-somes [41].

In the present study, the maximum cer-vical mucus sperm penetration distance(mm) value was observed by sperm ofthe 35 ppm Zn propionate supplementedgroup of bulls followed by 70 ppm and35 ppm Zn sulfate supplemented groupsand the lowest penetration distance wasobserved in the control group of bulls.Since no report is available in the liter-ature on the effect of supplemental zincon BCMPT, the present results could notbe compared. Improved values of BCMPTin Zn-supplemented groups may be due toimproved motility and livability percentageof sperm in the Zn-supplemented groups,since sperm the penetration depends on thenumber of motile and viable sperm [42].Higher intact acrosome percentage in Zn-supplemented groups might also accountfor it, since it helps in the penetration ofsperm in the cervical mucus [43]. One ofthe most important factors influencing thesperm penetration through the cervical mu-cus is the presence of anti-sperm antibod-ies in seminal plasma, which reduces nor-mal progression of sperm through cervicalmucus. Kramer and Jager [44] demon-strated that Zn- reduces the level of anti-sperm antibody in the semen, which maybe implicated in the improved penetrationvalues in Zn-supplemented groups in thepresent study.

The result of HOSST revealed thatthe sperm of Zn-supplemented groupsof bulls had higher tail swelling, afterhypo-osmotic treatment, as compared tothe control group of bulls. The resultswere in agreement with those of previ-ous researchers who observed significantincrease in HOSST response by supple-menting Zn in the diet of men [34] andsheep [5]. HOSST measures single fac-tor i.e. membrane integrity and Zn hasbeen reported to elicit membrane stabiliz-ing action by interacting with some func-tional group of the intrinsic component of

sperm membrane. It refers to the forma-tion of stable mercapeptides by reactingwith the -SH group of membrane pro-tein, which changes fluidity and stabilizesthe membrane [32]. Improved HOSST re-sponse may be attributed to increasedmotility and livability of sperm in Zn-supplemented groups. It is a known factthat sperm motility and livability is de-pendent upon membrane transport and itfinds support from the results of the presentstudy, where higher motility, livability andHOSST responses were recorded in all Zn-supplemented groups.

In the present study, a highly signif-icant (P < 0.01) increase in alkalinephosphatase activity was observed in Zn-supplemented groups as compared to thecontrol. Though, no report is availablein the literature for the comparison ofthe results of the present study, our find-ings are indirectly supported by reportsin which alkaline phosphatase activity inserum was greatly reduced in zinc defi-cient animals [45]. The increased alkalinephosphatase activity by Zn supplementa-tion may be due to the fact that, it is a Zndependent metalloenzyme and requires Znions not only as an integral part of theircatalytic apparatus but also as a structurestabilizing factor [39].

Significantly (P < 0.01) higher val-ues of acid phosphatase activity in seminalplasma were observed in Zn-supplementedgroups as compared to the control. Our re-sults are in agreement with those of earlierworkers [46] who observed increased acidphosphatase activity in men supplementedwith Zn in their diet. Acid phosphatase isa specific secretary product of the prostategland. The main source of Zn in semenis the prostate gland and it is requiredfor normal functioning of the prostate, soan improved value of acid phosphatase inseminal plasma might be due to the stimu-latory action of Zn on the prostate gland.

The mean GOT and GPT activity inseminal plasma of different groups of

Effect of zinc supplementation on semen quality 673

crossbred bulls revealed significantly (P <0.05) lower values in Zn-supplementedgroups as compared to the control group.It is mainly attributed to anti-oxidant andmembrane stabilizing action of Zn, whichcauses lesser release of these enzymes inseminal plasma.

The mean blood serum testos-terone concentration (ng.mL−1) inZn-supplemented groups was signifi-cantly (P < 0.05) higher than the controlgroup of bulls. The results of the presentstudy are in close agreement in man [47]and the rabbit [33]. However, no sig-nificant difference in testosterone levelswas observed by Zn supplementation inthe rabbit [25], man [34] and crossbredbulls [48], which may be due to speciesvariation, different duration and level ofzinc supplementation. Improved values oftestosterone in Zn-supplemented groupsmight be due to stimulatory effect of zincon testicular steroidogenesis [49], since Znaffects testicular functions by activatingthe adenyl cyclase system, which stimu-lates testosterone synthesis [33]. It wasreported that Zn stimulates Leydig cells ofthe testis and enhances the production oftestosterone [49] since Zn is an essentialcomponent of protein involved in synthesisand secretion of testosterone.

5. CONCLUSION

It may be concluded that supplemen-tation of Zn in the diet of crossbredcattle bulls improved semen quality interms of quantitative and qualitative char-acteristics of semen, as compared to thenon-supplemented control group, however,the organic form of Zn (Zn propionate)showed a better response in improvingsperm per ejaculate, mass motility andsemen fertility test like bovine cervicalmucus penetration, as compared to the in-organic form of Zn (Zn sulfate) at the sameand higher level of supplementation.

ACKNOWLEDGEMENTS

The authors are grateful to the Director ofthe Indian Veterinary Research Institute, Izat-nagar for providing the necessary facilities forcarrying out this research work. The first authoris thankful to the Indian Council of Agricul-tural Research, Krishi Bhawan, New Delhi forproviding financial assistance in the form ofa junior research fellowship. We are thankfulto M/s Kemin, Chennai for providing a freesample of zinc propionate for carrying out thisstudy. This work was carried out under AP CessScheme of the Indian Council of AgriculturalResearch, Krishi Bhawan, New Delhi.

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