Observations on artificial insemination of sheep with fresh and stored semen

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Understepomt Joumal of Feterinary Science and Animallnd1lstry, Volume 16, 1\'umhers 1 and 2, Jonum·y and April, 1941.

Printed in the C11ion of South Africa by the Gm·ernmcnt Printer, Pretoria.

Observations on Artificial Insemination of Shee p w ith Fre sh and Stored Semen .

By JOHK QUI~LAK ancl H . .P. STEYK, Section of Surgery, Obstetrics and Sex .Physiology, and D. DE YOS, Radiographer,

Onderstepoort.

'l'HE economic pos:;ibilities of artificial insemination in the production of livestock ha Ye stimulated intensive resean:h throughout the world during the past ten year;;. At first researches into artificial insemination were mostly confined to workers in Soviet Russia, where the urgency for the rehabilitation of livestock, subsequent to the termination of the Hll 4-1918 war, suggesteu the necessity for the utmost use of sires IYith <lesirable bloo<l. Artificial insemination, if successful, would extend the use of proYell sires considerably, and it would solve, to a great extent, the prohh'm of mpid rehabilitation of the livestock indu;;try 11·ith ~-hieh SoYiet Russia was faced.

'l'he Soviet Government founded a Central Experimental Breeding Station at }IoscO\Y iu 1919, and intensive study of the possibilities of artificial iusemination IYas begun in 1923 under the direction of Prof. E. I. Iva nov.

'l'he early publi<'ations of the \YOl"ken; at the Institute for Artificial lnselllination, at }[oscow, stimulatecl n·search whi<'h ~·as

taken up rapidly by most countries which were interested in the improvement of livestock. These researches have made considerable progress; so much so that artificial iusemination in the produetion of livestock has long since passed the experimental stage. The technique of the operation has been so perfected that the resultant coneeptions, \Yhen fre,;h semen is 11Sef1, is comparable IYith that following normal copulation. Artifi<·ial insemination appears to have become a routine practice in the production of livestock in Soviet Russin, where thousands of animals are inseminated annually.

It is claimed that there has been a rapid improvement in the livestock of that country since its introduction. Goetze (1939), however, points out that the visualisation in this direction has not nearly been reached. Artificial insemination has been successfully employed in the livestock industry in the United States of America, Italy, Holland, Denmark, Australia, Kenya, and to a limited extent in South Africa. Its chief adaption in most countries would appear to be to mares and cows. Ho~·ever, in sheep breeding countries it will be found eminently suitable for reproduction should the indica- tions for its employment out,reigh the contra-indication,;.

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ARTil'ICIAL INSEMINATIOK OF SHEEP.

Recent researches have been practically confined to improvement m the methods of obtaining semen, its storage, and its transport.

Observations on artificial insemination, especially with sheep and horses, have been made in this country since 1932. Quinlan, Mare and Claassens (1936) published a preliminary report on their observations with fresh and stored semen in merino ewes. Quinlan (193()). amplified the previous report in an unpublished paper read at a joint meeting of the )fedical Faculty of the Witwatersrand U ni versi ty and the Veterinary Facttl ty of the Pretoria U ni versi ty held at Onderstepoort. It has become a routine procedure in the Government flocks of large-tailed sheep, Ronderib Afrika11er and J:Carakul, to use artificial insemination owing to the difficulty which some rams find in serving ewes with the extreme tail-deYelopment peculiar to these breeds (Quinlan, Claassens, Bonsma and Rose, 19:39, Engela, 1940). The results ohbiued, ·when fresh semen is use<l, compare favourably with those foll(m·ing controlled mating with

"hand-service'': 47·5 per <:eut. of pregnancies were obtained when using fresh semen, as compared with 57· 5 per cent. by "hand service ". Only on insemination an(l one servi<·e wa:-; allowed <luring a single oestrous period. w-ith semen stored for :l hours at 8° C. to 13° C., 33t per cellt. peregnancies \Yere obtained. Ko pregnancies

\Yere obtained '"ith semen stored for G, 9, 12, 24 and 105 hours.

These results compare very unfavourably with the results obtained by other workers. However, it must be pointed out that only one insemination was made at a single oestrous period. No doubt the results would haYe been better if two inseminations had been given during oestrus, and if the operation had been repeate<~

in cases of oestrous recurrence.

The time of insemination during oestrus is important as it is

<lesirable that highly Yital spermi1to:wa should be present in the Fallopian tubes awaiting the arrival of an available ovum. Quinlan, Mare and Rou:s: (1932) obtained satisfactory results in merino ewes by normal matings up to the 33rd hour following the onset of oestruK . .Matings at the 36th, 39th and 42nd hour resulted in only 50 per cent.

of pregnancies, while only one pregnancy resulted from the matings at the 45th hour. Kelley (1937) found that spermatozoa, even from the most fertile rams, did not remain fertile in the genital tract of the evre for longer than ~l4 hour;;. Quinlan and Mare (1931) estimate<l that ovulation occurred in the merino ewe about the thirty-sixth to the fortieth hour following the onset of oestrus. 'l'herefore if sheep

;:n'e mated illunediately they sho"· oestrus the spennatozna must survive at least thirty-six to forty hours. However, it must be indicated that spermatozoa handled as for artificial insemination may have a lowered vitality in the female genitalia and consequently a lessened impregnating r-apacity.

The possible influence of copulation in hastenin"' ovulation can be . overlooked in Quinlan and Mare's experiments: as· the , sheep which were slaughtered to observe the time of ovulation had copulated with vasectomised teasers. It is not known to the authors whether obsenrations have been made Oll the influence o£ coitus on the time

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J. QUil'ILAN, ll. I'. STEYJ'I AND D. DEVOS.

of oYulation following the onset of oestrus in sheep, but recent vYork llone on heifer~ hy Quiubn, Bi;:sr.;bop anll A(lelaar (1941) has shown that r.;oitm; probably hastens oYulation.

Kardymovic (1937) says that about 20 hours from the onset of oestrus is the most favourable time for the insemination of merino sheep. \Vinters et alia (1938) give the optimum time as about 10 hocll'll frum the termination of oestrus and suggest bYo inseminatioils during one heat period to raise the percentage of pregnancies.

Buchannn-Smith (19:'l9) recommends insemination tlmanls the end of oestrus aml suggests the seconrl day as the optimum time. Since the exact time of on1la tion is not kno\\·u in the different breeds of shee11 the necessity for further study on sex physiology is pointed out bv Phillips (1939). This hiatus in the knowledge of the physiolog.v of the genitalia has been filled in under the enYironmental conditions preYailing in certain sheep breeding areas in South Africa, and the results will form the subject of an early report.

The effectiveness of repeated insemination of sheep during one oestrous period has been sho,,·n hy A \Tamov (1907), Perep:on (193o) ctllrl Gan·iloY (HJ:H). \Yhen the iutenal between insemination;;

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24 holll'S the land~ing 1·ate was inc1·eased by 11·7 and 14·1 per eent UavriloY Rug·g-est;; that the second imemiuation should he carrierl out later than 12 ho:us after the first. Pe1·egon recommenrls f.\yo inseminations at intervals of 20 to 30 hours.

The resultim;· rercentage of pregnancies is easily iuftuencerl by environmental facton; during storage. One of the most important of these fadoni is temperature. Spennato~oa tlo .uot surviYe long at room tnHuerabll'e in this country, "·here shade tempe1·atures of a roull(l 90° F. a llfl oYer are frequent l_v reeonl erl hehYeen Sept em her anrl April.

It

has been ohserYed that a temperature 11ot far above 0° C. is the optimnm for storage of m-ine spermatozoa. Storage, even for short periods, at room temperatures, rerlnces the impreg- nati n ·~, ca par-i t.y of ~penn ;ttozoa eYen t hm1gh they retain adiYe motility. Attempts at storage at tem1Jerahnes of bebYeen 10° C. to 12° C. haYe failed to maintain Rperm acti,·ity for longer than 6 rlays.

The sperm atnr.oa from the same rams ''"hen Ptorerl at tem pern t.u res l:.etwPen 2° C. to 4° C. ha,-e sho\Yll actiYity up to 34, 46 49, :)0, 61, f'J4, GG, 72 and 82 r1a:n;. Tn oue case longeYity up to 100 days "·as

n~c-orclecl.

Dnri ng- recent yen rs ,-er~- exteusiYe literatnre has appearer1 ⁰^1.

the inftnence of temperat11re anrl other environmental factors on the Yib lity of spermator.oa. Bonadonna (1939), \Yho sent a qnestionaire, req uesti n ^Q' inform n tiou, to the \•:orke1·s in every r:onn hv where PXperimen ts Oll artificial insemin a tj On \Yere being' Conflucteri, quotes the principal \Yorks relating to storage and shipment, anrl no attempt is made to give a complete reYie"- of all relevant literature in this report.

Bonadonna indicates that the optimum ronditions for the prolongerl storage of semen indurle lo"· temperature, the re(lnction of s11ermatomal movement, the maintenanre of sufficient amounts of nourishing materia 1 (glucose) nnd of material able to neutralise, as 11111Ch as possible, the toxic efferts of the prorlncts of metabolism, th1w effecting· a balanl'e of the H-ion concentration.

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ARTIFICIAL INSEJil:I>ATION 01-' SHELl'.

The pH of normal ovine semen, tested within half an hour after ejaculation, is acid. The pH range lies n·ithin 6·3 to 5·.S . .. When fresh semen is stored at roolll temperahue the1·e is an increasing acid reaction. This change is apparently due to the pr0ducts of metabolism, because it is less rapid wheJL the spprn1 activity is lowered by reduction in temperature. Our tests haYe been made ,,·ith a Beckmann pH meter with glass electrodes. The following is a typical example of the observations made. The semen of ram 45106 just after ejaculation on 17.5.40, slwwecl a pH of (i·3; on 2-L0.40 it 11·a,; 5·8, and after 32 days it 1n1s 4 · 4 "·ith ;;perm motility still fairly actiYe. According to vVebster (1938) the semen of highly fertile rams is acid with Yery rare exceptions. vVhen testing the semen of rams for fertility by means of artificial insPmination the most sueeessful results \Yere obtained 11·ith semen lun·ing a pH range hebYeen 0 · 00

•111(1 6·50. A Yery slight acidity indicated lo11· fertility, while alkaline samples were from in fertile rams. :U oskoitis (1934) states that the best pH for dilutors is 7·3 to 7·5, while Lardy and Phillips (1939) give the optimum-pH for storage as ()·72 to ()·80. The experience under the em·irolllnent preYailing at Onclerstepoort j,;

that there is a gradual inc1·eu;;ing- acidity with ston!cl normal ru111

semen until active motility of the spermatozoa is reduced by eooling in the refrigerator.

Fig. I.- Photograph of ~orne of the menno ram~ u~ed.

The method of obtaining· semen is also ll!Ost important for the optimum 'longeYity in storage. The utmost care must he taken to ensure absence of c-ontamination. The method at prt>sent most favoured in sheep iR the a1·tificial Yagina, ''"hieh for practical purposes appears to supply :dl rpq ui rcments.

\Villiams uncl Sa1·age (1927) lw,·e remarke(l npou the 1·eststance of spermatozoa to cleeomposi ti on. In one iDstance the longevit.Y of spermatozoa in coJJtaminated selllen "·as 43 clays. Baker (1929, 1931, 1931, 19;~2) thinlis that eontnmination imposes a definite but nDt very marke(1 limitaion on the 1oltgeYity of :-;pennatozoa in vitro.

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J. UlTKLAN, H. 1'. S'l'EY"' A~D D. DE VOS.

In our experiments an increase in the bacterial content of stored semen, a'lthough not incompatible "·ith sperm actiYity, has quite definitely limited longeYity "·hen eompcn·ed ,,·ith semeu in whieh bacterial life "·as not so actiYe.

In our obsen·ations the ~l·men has been stored b:y means of the Cambridge method de:seribe(l by \Yalton (Hl:Hi). AJtemlJb haYe beeu made to correlate longe,·ity in vitro with the fertilising po,:·er of svermatozoa. During the~e studies it has been noted that actl ve motility' is uo c-riterion in tletermining the fertilising pO\Yer of storetl ile1ue11. Ilahilmlliu (HJ:JS) lws matle similar obserYations. The longest period cluri11g "·hich spermatozoa haYe rem a inetl motilf' wa,;

100 clays. SeYeral specimens \YCle found actiYe up to forty-six to fifty-four day,;. Oviue spermatozoa in Yitro have been obsened by workers in other countries to be acti ,.e after seYetal days: Lebecleva (193-t) 29 days, Gnnn (1936) 28 days, Bonadonna (1939) 569~ hours.

F'ig. 2.- Photograph ~ho,,·iug the type of merino ewe used: Taken just after shearing.

The results of nrtifi('ial inRemiu:dion \Ytth storerl spenn, in our luuuL, haYe been (lisappoinfing· since highl,v ndi,·e, mo1·pholog·ically ulU·hanged spermatozoa haYe failed to impregnate after 72 hours. Uther \Yorkers h<.tYe hnd more encouraging result:;: Goetze (1!)33)

\\·as succes,o;ful \Yith oYi11e semen stored fell' 9-± hours, Phillips, Schott :lllcl Gilclo,,· (Hr18) J 15 ho1n·s, B01wclonna (J!J3!J) 5:; to GU hour:;.

Chum (1U;~G) ''"as stl<'cPso;ful \Yith semen stored tor 24 l1mn;,; at. -5° C., 6 hours np to 18° C. aud 2 l10nrs aL ]8° C., fo:Ilo\Yecl b.v 30 hours at G° C. (total ;J2 hour~). \\'inters d alia (1U;38) .~tate that at Minnesota seYeral ewes h:ne been successfully int>eminaterl "·ith :"penn four aml fin) clays olcl :md t\\'o shee1J \\·ifh sperm stored for six days .

• \ttempts to hans]Jort "lorerl semeu haYe been suecessfully nr;complishe(l, but to a limite(l extent. Sue('essf ll tnnlt>]Jortatioll of sheep semen bas been Clone by \Valton and PrawochPnski (1936). Slwe11 semen ,,·a~;; tlent from Cambridge to \Var~a1r. l'm) of fiYe

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AllTU'ICIAL INSEl\liNATlO~ 01' SHEEP.

::;heep artificially inseminated became pregnant. The rluration of storage \Yas 51 hours. Bonadonna (1038) ~'Jcceecled in impregnating four out of seYen e"·e,; 11·ith ::;ewen sent hom ::\lilan to )lonza, the journey taking senn hours. Phillips (1939) (the \York "·as done co-operatively by Dr. H. \V. Phillips, Dr. E. )l, GildmY, Dr. R. I.

Schott and Dr. C. E. Terrill) re<·ords the hanRporiation of ram seme11 b·y air express hom Dubois, l<laho to ::\losco"·, I<laho (700 m i:les), HeltsYille, Maryland to ::\Iose<m (2600 miles), and in both rliredions behYeeu BeltsYille an<l Dnhois (2250 mile:;). The time in transit

()(~tupied 19 to 74 hour,;. Of :J()g in;;eminations 31 resulted in }Jl'eg- nancies. The ol<lest St'JIJPil use<l successfullv 'n1s lJ Fi hours.

K 1llesen ko (190 7) .~uccessf ully trans} 1orterl st•meJJ :JOO Kilometres. ,'\.ll the semen nserl wn s ¹¹ot ira ns vorterl, but hi:> resulting fertility

i~. remarkable: 15,000 sh<>ep "·ere iuseminnied "·ith the semen obtained from one ralll a:l(l thP pen:entage of barrenness "·as not more than 1-:2. Phillip;;; sayr; that his 1·esults inlli('ate that the lJracticahle use of l<Jlll c;enJPil, "·here storage periods of one clay ol' more a1·e ner·efisar_,., ,,·ill lw limited until techniqtH' fo1· stmage i:-;

uuproYe<l. The methods <'lll}Jloyt>rl in pa<"king for transport are morb or les~; i:iiltlilar to that reeoJJJlllelllled ll\- \\'alton aurl l'r;J\\'O<"ll<'llSkl (19;1G). The semen is put into uano,~· test tnb<·s, cO\·m·ed "ith a laye1· of sterile liquirl paraffin and corked, uo air ;;p;t('e being left. This tube is placerl in a ('ontainer to prewnt hrenkiug. The r-on- taiuer i:> then nut into a the1mos fln;.;k fi I led "·ith ice and \\·;d.er.

13oua(louJW (l9:.f8) sugg-ests the nrlclitioH of a gluco-vhospl1ate solution (0·1 to 0·2 <·.c. for rar-h <".<·. of semen) for sl1eep semen.

Sne<"e~,.;ful attempis han' heuu ;nad<' tu iJanspod g·pi;J1inis\'d

;;petlllatozua in f'apsules: ~JilonuJoY, 1\ajnrnyi, Sin)korj an<1 3fnlohm· (1937) and HazmnoY (193S).

:\o aitemptii haYt> berH ma(lt' to irau~port OYlne spennatoiiiJ<J £01 nse in ;1rti:ficial insem.iJJation in this eountry.

In our ob;;~;;n·.aiions it has been nott>1l that oul~· highly actiYe

~pennatozoa will establisl1 pregn<\ll('.\'. li has been ;;nggesterl that stored spermatozoa usrrl for artifi('inl i11seL1Jination may prorlu('e weak pfffipring (Bon<tclonna, lD:-m). On<· <lPfonne<l lmnb "·:1s hol'll in our experiments (see Tahlt> ±) bnt <Ill others "·err health~- b mhs, of Jlonnal "·eight anrl their post-natal <le1·elopn1ent \YU:i ex<"ellent- (Fig. 3).

It appears highly probable that ,;iore<l spPnnatowa ('apahle of fertilisation will produce healthy offspring. llonarlonna (] 9a8) J·ecommencls, from a practical point of Yie"·, that oYiue sperm shmd(l not be storecl for longer than ;{Q t-o 50 hom·s prior to 11Se. Thi~

recommendatioJJ was marle afte1· a re,-ie,,· of the most importnt1t publications published up to 1039.

'l'he methods of co'lleeting ram semen for artifical insemination have been so frequently ch~sc1·iberl '"ithin recent years tl1at it is ttnnecessary to repeat them: Anllerson Hl07, \VinterR et al. 1938, Walton 1938, Webster 1938, Gunn 183G, Goetze 1939, Brady and Gildow 19;l9 anrl other;;. Most \Hlrkers are in agreement that rollection by means of the artificial vagina is best suited for the

n~m. It inYohefi little bb01.n a11el trouble and a specimen free from 2GS

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,T. Q"C'IKLAN, H. P. STEY:N AND D. DE VOS.

(:ontall!ination can lie obtained. Brady ancl Gilclow (19:39) say that the artificial Yagiua is thP most sntisfadory for rapid aml eas~·

L'Ollection. However, electric stimulation is most useful when semen has to he <·ollede<l hom seHral ran1s or \Yheu a ra111 will not or cannot copulate \Yith the artificial Yagina. A most suitable eolledor is that described by '\Valton (1938). The pattern clescriberl hy

Fig. 3.- Photograph showing some of tbe lambs born from experimental e\\'es.

Fig. 4.-Photogmph of ewe in senicc uail.

\:Vebster (1938) is abo Yery suitable, b11t \Yith \Valton's artificial vagina there is 110 uec·essit~· for insertion into the Yagina of the f'We.

Hamil trained to ~erve sheep in a ;;e1·Yice bail, as shmvn iu Fig. 4, will nse the artificial ngiua '"ithout difficulty. Hoehn (1 934) indicate~

thnt the temperature of the artificial Yag-ina must he maintained at

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ARTIFICIAL INSEMINATIOK OF SHEEP.

approximately 40- 41° C. to ensure ejaculation. Brady and Gildow (1939) and Goetze (1939) have also made similar observations.

Walton (1938) says that the temperature of the artificial vagina at the moment of collection should be between 40° C. and 45° C. At a lower temperature ejaculation may not take place, while a higher temperature may be detrimental to the spermatozoa. vVhen using semen obtained by electric stimulation, as suggested by Gunn (1936), the resultant conceptions do not appear to be so satisfactory as those obtained by collection in the artificial vagina. Kelley (1937) had only twelve pregnancies from fifty-five sheep when using semen obtained by electric stimulation, as compared with f0rty-nine out of seventy-nine " hand served ", and seventy-two out of ninety-three

"paddock " mated. Kelley, however, points out that his e\Yes were not inseminated under ideal conditions.

~L-\TERIAL AND METHOD.

Two hundred young sexually mature merino ewes between the ages of

li

and ;3 years, were placecl under observation for oestrus.

They were tested for oestrus daily at 8.30 a.m. hy using vasectomised teasers. 'rhe sheep 'Yere run under " dry lot " conditions in two kraals, each measuring 75 yards by 60 yards; a wood and iron shelter was provi(1ecl in each kraal. Fresh water from the tap was always available. The ration consisted of crushed yellow maize, 3/4lb.; Cl'Ushed oats,

t

lb.; lucerne and veld hay ad l-ib.; green food, either lucerne or barley, ad lib.; bone meal, } oz:. and salt

k

011.

'rhe concentrate ration was given in equal amounts twice daily.

Green food >vas feel once daily in the forenoon. 'l'he hay was always available.

All the Rheep had been snbjected to routine vaccination against Blackleg, Anthrax and Bluetongue. At the commencement of the observation the sheep were in moderate condition, but they improved rapiclly an<l maintained pxcellent con clition throug-hout (Fig. 2).

They caniecl approximately six m.onths wool "·hen the ohservationR began.

All ewes were marked over the rump witl1 the sequence numbers, and their group number was marked over the hack. The laboratory registration number, which was recorded by ear-tagging, aeted as a eheck. Nine vasectomised teasers were used for testing. These were half-bi·ecl vVelsh-Mountain X Ronderib Afrikaners, a cross which makes an excellent teaser.

Testing for oestrus vvac; clone daily, except Sunday, at. 8.30 a.m.

The sheep were driven into a collecting pen, and then passed in hatches of about twenty through a teasing pen in which four or five vasectomised teasers were placed. The teasers were rested periodically when it was considered necessary. Ewes showing oestrus 1Yere taken out and placed in a small kraal, which contained a brick-walled shelter with a galvanised iron roof, where the inseminations were earriecl out. Only sheep which accepted coitus willingly were considered as being in oestrus. All inseminations or normal matings, in the case of the control group, were done within six hours from t1)e time of testing.

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lt i:o 110i possible (u t•::;(im;t(P 11·i!lt any degret' of ;~<-cur;w_,. !he

<huaLion of oestrus at i he ti.me of iust•minahon OJ' ttortttal m;Jting.

lt m;ty han• begun <~L any time 11·ii.ltin i.lte pre1·ious i.ll'l'td.l-futtt hours.·

.\.f(er ilbl'lllinaiiou ot· nonnal lll:thttg· ⁽^Jt^p e11es ll'l'l'e re(uull•d to tlte original eamp "·!ten-• they \ll't'e suh;uitted to tht· dail.1 routine testing uni.il they \Yt're <·onsidt'l'P<l]Jregttattt or had lll'.<'n inst•tnitJ;d<·d or mater1 for ihe ihirc1 hlllP ;1( ~uct·Ps~i,·e recurTt'llCPs of OP~i t·us.

Tltt' t'\\·ps 11·<,~1<' rli1·i·h·rl into ien gTO<lps of (ll·<·tlt\· she<'Jl t'at·il:

(;toup I \l;t:' a r·otttrol gmup itt whir·lt tt<trntaJ m:dit1g· 11·;1S ranit•d utti: (;;·oup ~! 11·as ittsetttinated 11·iilt +rrsh S<'llll'n 11·iihitt fil'tt'l'lt miuuit•s follo11·iHg e.iar·nhd io11. Tlil' l'<'ll1aining ::;elt'll gnllt]Js <I to <J.

\l'l~l't' in.-;etniu:tiP<l \Yith storetl :;etltPil, ke11L in Yiho a( " iPllljlPI';d.ure ol' lwill.<'l'll ~° C. to -1° ('. for (i, 12. lc, :2-1·, -18. 1'2 <~tHI !Hi !tonrf'. Ct·oup 111 1\';J,; iusPI1Jitt:tied 11·ii·h ,;t'lJI<'I\ shll'P(l from .-J to II d;t\S. 'J'hP

<bb ·;tt'l' n·r·onlcd i11 Table::; 1-10. TLirteeJJ n,<•rin(• r:lllh II.'L'l'l' used for tttcti ing ;\tiel <Is tlunors for obiaitt ittg ,.;(~11\l'll fo1· itl:'etninalioH. ]'rior to the <'OJllttl<'ll('l'tllPid· ol: the ol:,en·;dJot\..; iltest' t·;\tll..;' "l'J\1('11

<\1\tl spt·rn];llozo;\ h;~d hceu le.-;terl ;tttd ]'Jol·t·d io Le plt\·:-iil'<~ll.'· a11d llll!rJllwlogit·::tlh tJUJJU;d. TJ1e semen 11·;~..; r·ollcdl'd .i11 <Itt al'tifici;d Y;lg·itw (Holhorn [JJsirutnt•nt Coy. i,l)ll'. ;Js n•cotltllll:'tllil'(l 1,,~· \\';iliott.

l!J.J(J). Prior to ll:-il' thtcl ntld:er litllllg' \\·;1..; ,,.;tslwll 11illt ..;oal' ;1nrl

\1';\tet·. It 11·a:e thc·n t·il:.,ed tlwroug-IJI1· 11·ith ntltllilJg· \\<Jter to t<'ttto~t·

all trac<'~ oi' ..;oap and tit1ally ·'''·aidtt•d 11·ith (;.-, per t'Ptlt. ;~kohol ;tttd ,J!J.o,, eel io d1 \ lwfrll e l:eing- used.

Jl.efore li"L' II'HJ'Ill 11·all'r 11·;~..; phtTll 111 tbe j<~ckd "' ilt;d lilt•

tem]JL'l·;dun· "",; ;tbollt lll-1:⁰ 1:. io 10,-,o F. ),ir 11·;1s pulllllt'd in io l1ring il np tn t lJP requirPd pl<',,.,lirP. If the pres.-;1ne of il1e ;trlifici:d Y<lg'ilt;t 1\';JS 1\0t. ndj1bietl Cill'l'Pf'ih·. m· jf tlll' tl'l11jlE'l';JiUJ'p \\';\~ (oo hi!._!·h ur too lo11 l'.iaeulatiot\ di1l noi u..;n;ill.~· tn],p pbl't'. Botb -f::tdnr..; 1';\Jl hl' conttollt·cl qui<·kl_,. ~\tu1 ;tc·<·UJ<-lt~l.'· h.\· ~111 expericllf'Pd \\-o1·!..:v1·.

\\'hPJ\ •·olll'ciitlg· Sl'lllt'll 11\· Jllt'<lll1i of (!J,. ;tdifil'i;tl ,·agttl;J it t..;

l\l'l'('-;-;;n\· io li;)Yl' till' PII'E' \\·cJ[ l'Oil( t·ollt•d in ^<I 1i8ll·i<·<• h:til, 1iUl'iJ ^;)b tlt;d· slJo·ll.ll in Fi;.:. J. 'l'lt<' py;e sbtt<L firt1tk ~l'C1li'E'd 11·iih ihl' hind (lll<ll'i<'l'' ..;ttfhl'ir•tiil\· dv;~t· !'or il11• l<llll io 1\\olltli.

\\'ht•Jt ir<~initlg r;\111.' in lhl' t11<' :p·tifit·i;d Yag·i11;1 it i:; a<hisahle lo <Jllo11 iht'l\1 lo l'lljlltlaie tiOl'lltally 11·iih l'll·e,_; ..;lwll·ittg· OI'Sirus. \\hil'h

;Jtl' <·onfined itt the o;en·il'<' l::1il. \\'il.h lt::tineti 1·am:; atiV (~''.<' •·an lit' us<'rl lor l·ollediot\ ll·itl'iltt•r slH· i.' i 11 <J<•si t'tt..; 01· t\IJI.

Afic>r Listening tlte ell<· in tltt' spn itT h1il !Ltt' :Jriifi,·i;il Y;~g·ttl<~

\Y;\~ lJrP)I:llT<l ·fm· 11SI'. Tl•t' ran1 ""·' tll<'ll ;tdtnitte'l io lht> l1oUsP.

HP 1t~n;~lly tri<·cl to tllllttni imm<-'<li:d·eh·. \Yht>lt monniiu.g· lhe )ll'lli>

'""" deflt·l'iP<l into the ;lriifici;Jl 1ag·ina \1 hich 11·;1.s phretl ;llongsid,, tl1e ripJli i1auk of l'lll' e11·e, ~~:i sltO\YtJ in Fig. 5. The pe11is IYas not toudecl 11·ith ihe h;Jncl. the ddll'dion heinp: rlone b,1· p:r:tspiug tltL' sl1e::tih . . \fier ej<~clilatiou tlt<• presc'tll't> in tl1c artifici;tl Yagina "·a,.c immedi;Jtel_,. n·le;~'ied b~· opc·ning thC' ~top-rock. Tt "·;1s lwld uprip:lti

!'0

n ••

t iltP ~elllE'Il ]';\[\ into He gla.'·" contaiJH'l'.

')~

- 1 '?I J

(10)

AltTIFlCUL TXSE~U ~ATJOS OF SUBEr.

If the ,.;emen 11·a~ to hP llsPd for i lllllJed iate iuselnin;ltion it was diluted in the l'Ontainer and "·ithdn111·n from there. If it ,,·n:;

to he stored it was remowrl hom thE· C'Ontainer with a sterile pipette and transferrc:d to llUJTO\Y test tuLc•s, J iJH:hes hy i} im:h, anc1 l'OYrrecl with sterile· mec1icinal liquid paraffin. This ill the treatment fm·

storage of semen rec·omnJPJJC1PCI h:v \Valton (l93G). ]t was then left to cool to l'OOJll temperatm·e for <Jhmn l\Yent.v minutes. 'rhe tubes

\YOre then placed in a ll'<ltPr l1nth <lt room telllporature nnd hansfel-rocl to the rpfrigerator.

Fig . . j.- Photogrnph of t·ant I'O!>Illating 11·ith till' arlirit'ial yagina.

The intlnenC'e of Yario11s tempenliure::; 011 the long-evity of the

;-;pennatozoa "·a:; triE'd. It \\'<JS found that the oplin1mn temperature for the preserYatiou of life ancl adiYit~· of ovine spermatozoa in ritl'o was between 2° ('. and -1° 0.

An exteusiYe ,.;eries o£ ol.J:;etYai,ion,.; \YOre made iu order to

<~scertain the optimum pnvi1·onmental eondit.iom; for the pre:;erYation of maximum actiYit.y aftr1· stOl'np;e, anc1 those nhoYe cle:;rrihed haYe given the most succ:essful results. B,y this metl10d live sperms were seen after a storage period of 100 days, anc1 seYt>ral spec:imens showed

:JCtivity for varying periods up to 82 days.

272 t

(11)

J. tJLT~\L.~X, II. l'. STEYK .U;-D D. DE YOS.

The :;emen when beiug removed hoJJt the rehigerator for use was allo'H'<l 1o ,.;tand for about twenty minute:> until room temperature wac; ngaiu <tttaiued. Before mixing the semen with the dilutor ture wa" Ltken that both "·ere of equal temperature.

The motility and sperm densit.\· of all specimens of semen used for artificial insemination \H're controlled, both at the time of collection :mel immediatel.\· prior to using-. Only spPcimens considered to be of high quality were retaine<l. 'l'he longeYii:v of spermatozoa in specintPllS whi<"h were not entirely used for insemination was almost invariably obsen·ed after tlwy were 1·eturnecl to the 1·efrigerator. 'l'lte semeH userl in g1:oup 2 ,,·as uot subjectecl to microscopic exanuna- tion lJl"ior to me as it ^\YU^il11serl immediately after collection and was kno,,·n to come from ltig·hl.Y fertile ntlllii \Yhi<"h "·e1·p ltSNl throug-hrmt the obsenations.

The ,;penn dilutor u,.;e<l ''"'t.~ m::ule UJJ accm<liug to thn formula of \Vinters et al. (JD38). The formnh is us follows:

Ulueosc 50·00 grams.

)

KH2PO., 0·32

"

N n2HPO" J :2H/) 1·54

"

J

pJ17.

Dist. H20 1000 c.r.

CnSO, Saturate.

The semen 11·us <lilute<l four times for each insemination innne<liaiel y hefo1·e using. The i nstrnments used \YeJ·e t l10se recom- mende<l l1;~· 'Valton ( l9;18) : The_'.· 11·ere purclt asP<l from the Holborn Instnm1e11t Coy. Ltd., London. The syringe ~~·~ts an ai.l glass t.nw fittwl to a Yulcanite nozr.l<~, 10 iJI(:bes lung. 'l.'l1e Yagina of tlte e1Ye was tlilated with a Yaginal speculum or a pyrex test tube,

i

inch in diameter and G inches long-, from whir-h the f'nd hacl been n'llloYP<l;

the J:ough edge was smoothed in a bunsen flame. The spE>cuhmt befm:c insertion 11·as lubricated ''"ith sterile liquid paruffin. It was inserted sufficiently cleep to exposE' the opening of the cervical c::~nal.

Illnntination of the intf'rior of the vagina was ohtaine<l by me:u1~

of an electric forehead latnp. The vulcanite nozzle of the syringe, nmtnining the desirl'd qmuttit.\· o£ diluted semen, was inserted into the PerYical canal to a depth of

a

to 4 mm. and the injection marle into the lumen. The quantity of diluted semen used was 0·2 cc. to 0·4 cc. 'rhe whole injection does not remain iJJ the eenical canal; a portion usually returns to the vaginu. \Vhen penehation of the ostium exten1um wa.s <lifficnlt the semen was cleposited at the opeui11g to the t·anal and on the pars vagina of the cen·i.x; this method proved equally successful as injection directly in to the cenical coanul.

During insemination it i" necessary that the ewe remains quiet. This ean be effected hy the use of an i11semination erate, :ts shown in Fig. G. This method is comfortable for the operator as the crate <:an be adjusted to the desired height. Ho\\·e,-er, it neceR.sitates raising the ewe i.nto the Prate for Parh insf'minati.m~.

273

(12)

ARTIFICIAL INSEMINATION OF SIJEL'l'.

i'lg. 6.- Phoi.og;raph of e"·e iu in>emination crate sho"·ing adu:li inseminatioo in standing position.

A simple and less laborious method of holding the e'"c is shmn1 in Fig. 7. The sheep is hell! in the clorsal or dor,;o-luteral vositiou on the~ sack placed on the grouurl. The opm·ator kiwels behind the ewe during the operation. By this metl10cl less assistance is ueeded

~.nd control is equally effectin.

Fig. 7.- Photograph of ewe being inseminated in dorso-lateral position.

274

(13)

J. QUINLAN, H. P. STEYN AND D. DEVOS.

After each insemination the speculum was cleaned and wiped with gauze soaked in 65 per cent. alcohol. It was allowed to dry before being used £or the next insemination. The outside o£ the vulcanite nozzle was similarly treated by wiping with gauze, moistened with 65 per cent. alcoho1, after which it was wiped off with gauze moistened with dilutor.

Sheep which did not become pregnant to the first mating or insemination were subjected to similar treatment when they again showed oestrus after the termination o£ an ovarian cycle. These s.heep were mated normally or inseminated three times in their respective group, once during ·each successive oestrus. In groups 8 and 9 some ewes were inseminated only once or twice., although they did not become pregnant. Failure to <;arry out the usual three inseminations was due to the fact that there was no semen o£ suitable age at the time oestrous recurrence appeared. After the third service or insemination, whether they became pregnant or not, the ewes were placed in a camp where they were kept under the closest observation for abortions. Towards the termination o:f the gestation period all ewes showing udder development were placed in a shelter where they were allowed to lamb. Records were kept o:f the percentage o£

conceptions £rom each-service or artificial insemination, the number . o:f services or inseminations required to establish pregnancy, the

duration o£ gestation and the sex and weight o£ the lamb.

The following Tables 1 to 10 show in summarised form the results obtained.

28

(14)

113 53353 8/2/40 45301 26/2/40 45129 21/6/40 45106

-

+

= l!'ertile.

Service.

+

14/ 7/40 Male .. 6! 149 l 104 55722 16/2/40 45301 6·99

- - - - - -

14/ 7/40 Male .. 7! 149 l 105 53172 17/2/40 45301 6·99

- - - - - -

14/ 7/40 Female 8 148 1 106 55881 17/2/40 45301 6·99

-

--·

- - -

15/ 7/40 Male .• 8! 149 l 100 55876 17/2/40 45301 6·99

-

- - - -

15/7/40 Male •. 8:} 149 1 (;)

...

---.__ ·--

s

* Dystokia : Lamb born dead during night. ~ Females ... 10 52·6 per cent. } A . ht 8 l" A t t' · d 149 2l d A b f · · t' 2 > Males...... 9 47. 3

per cent. verage we1g , u.; verage ges a ·lOU per10 , • ays; verage num er o 1nsemma 10ns, • ~Z NOTE:- Percentuge Conceptions for:-l'ta.m. lst Insemination, 30 per cent 45095 ... . 2nd Insemination, 35 per cent. 45103 ... . 3rd Insemination, 30 per cent. 45106 ........ . Non-pregnant, 5 per cent... 45129 ... . 45131 ... . 45135 ... .. 45219 ... . 45301 ... . 95 per cent. Ewes lambed. 45308 ......... . 5 per cent. did not conceive X ... .

Insemination.

+

l 4 3 1 3 2

7 5 7 4 1 1 19 20

Conceptions for dilut01· pH.:- 7·34 ........ . 7·21. .............. . 6·99 .......... . 6·9 ... . Conceptions.

+ -

3 7 l ll 12 5 -

-

19 20

+ =

Fertile •. = Not fertile.

~ "d 00

..,

~

z ~

~ ~ "l 0 00