U.S. patent application number 09/736841 was filed with the patent office on 2001-09-06 for (n-3) polyunsaturated fatty acid compositions.
Invention is credited to Noble, Raymond C., Surai, Peter.
Application Number | 20010020041 09/736841 |
Document ID | / |
Family ID | 26309607 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010020041 |
Kind Code |
A1 |
Surai, Peter ; et
al. |
September 6, 2001 |
(n-3) polyunsaturated fatty acid compositions
Abstract
Methods and compositions for improving the viability of sperm
are disclosed. The method involves adding (n-3) polyunsaturated
fatty acids to antioxidants and sperm or seminal fluid, or to the
diet of the animal producing the sperm. The compositions
incorporate (n-3) polyunsaturated fatty acids and antioxidants in a
feed supplement.
Inventors: |
Surai, Peter; (Ayr, GB)
; Noble, Raymond C.; (Ayr, GB) |
Correspondence
Address: |
HESLIN & ROTHENBERG, PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
|
Family ID: |
26309607 |
Appl. No.: |
09/736841 |
Filed: |
December 14, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09736841 |
Dec 14, 2000 |
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09214174 |
Feb 24, 1999 |
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6235783 |
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09214174 |
Feb 24, 1999 |
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PCT/GB97/01735 |
Jun 30, 1997 |
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Current U.S.
Class: |
514/560 ;
435/2 |
Current CPC
Class: |
A61K 31/202 20130101;
A61K 31/335 20130101 |
Class at
Publication: |
514/560 ;
435/2 |
International
Class: |
A61K 031/202 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 1996 |
GB |
9613754.2 |
Feb 26, 1998 |
GB |
9704018.2 |
Claims
1. The use of an antioxidant to enhance sperm function and/or
viability.
2. The use of a polyunsaturated fatty acid (PUFA) to enhance sperm
function and/or viability.
3. The use of an antioxidant accompanied by a PUFA to enhance sperm
function and/or viability.
4. The use claimed in any of the preceding claims, wherein the
antioxidant and/or the PUFA is administered to the animal producing
the sperm.
5. The use claimed in any of claim 1 to 3, wherein the antioxidant
and/or the PUFA is added to the sperm or to fluid surrounding the
sperm.
6. The use claimed in any of claims 2 to 5, wherein the PUFA is an
n-3 fatty acid.
7. The use claimed in any claims 1 and 4 to 6, wherein the
antioxidant is selected from vitamins, plant extracts and
carotenoids.
8. The use of a PUFA to enhance sperm function and/or viability in
avians, wherein the PUFA is an n-6 fatty acid.
9. A method of enhancing sperm function and/or viability,
comprising adding to the semen of an animal substantially
sperm-free seminal fluid containing an antioxidant and/or a
PUFA.
10. A method as claimed in claim 9, wherein said seminal fluid is
produced from the semen of another animal.
11. A method as claimed in claim 9 or 10, wherein said seminal
fluid is produced by removing sperm from the semen of an
animal.
12. A method as claimed in claim 9, 10 or 11, wherein the
antioxidant is selected from vitamins, plant extracts and
carotenoids.
13. A method as claimed in any of claims 9 to 12, wherein the PUFA
is an n-3 fatty acid.
14. A method as claimed in any of claims 9 to 13, wherein the semen
with the added seminal fluid is then placed in cryoscopic
storage.
15. A method as claimed in any of claims 9 to 14, wherein the semen
with the added seminal fluid is used for artificial insemination.
Description
[0001] This invention relates to improvement of male fertility.
[0002] All animal species' spermatozoa have high concentrations of
polyunsaturated phospholipids. In. mammalian species e.g. the bull,
boar, ram and man, the substantial level of polyunsaturates present
is characteristically dominated by docosahexaenoic acid (22:6,
n-3), a fatty acid of 22 carbon atoms in chain length, containing 6
double bonds in n-3 conformation and belonging to the
alpha-linolenic acid (18:3, n-3) series. Thus in the case of the
bovine, docosahexaenoic acid accounts for around 55% of the total
phospholipid fatty acids, with particular concentrations occurring
within the phosphatidyl ethanolamine and phosphatidyl choline
species. By contrast, avian spermatozoa exhibit in general very low
concentrations of docosahexaenoic acid and acids of the n-3 series
but this is compensated for by the presence of substantial
concentrations within the phospholipids of polyunsaturated fatty
acids having chain lengths of 20 and 22 carbon atoms, containing 4
double bonds in n-6 conformation and belonging to the linoleic acid
(18:2, n-6) series; these are arachidonic (20:4, n-6) and
docosatetraenoic acid (22:4, n-6) respectively.
[0003] The lipid composition of the spermatozoan membrane may be a
major determinant of motility, cold sensitivity and a wide
selection of factors associated with overall viability within fresh
ejaculates or stored ejaculates maintained at -196.degree. C. for
artificial insemination.
[0004] According to the present invention there is provided an
antioxidant to enhance sperm function and/or viability.
[0005] Further according to the present invention there is provided
a polyunsaturated fatty acid (PUFA) to enhance sperm function
and/or viability.
[0006] Still further according to the present invention there is
provided an antioxidant accompanied by a PUFA to enhance sperm
function and/or viability.
[0007] The antioxidant and/or PUFA may be administered to the
animal producing the sperm, for example in its diet, or
intravenously or intramuscularly, or may be added to the sperm or
to fluid surrounding the sperm.
[0008] Preferably the antioxidant is selected from vitamins, plant
extracts and carotenoids.
[0009] Preferably the PUFA is an n-3 fatty acid, for example
docosahexaenoic acid (DHA) or another member of the alpha-linolenic
acid (18:3, n-3) series.
[0010] In a further aspect, the present invention provides a method
of enhancing sperm function and/or viability, comprising adding to
the semen of an animal substantially sperm-free seminal fluid
containing an antioxidant and/or a PUFA.
[0011] The seminal fluid is preferably produced from the semen of
another animal which may have been vasectomised or from whose semen
sperm has been removed.
[0012] The mixture of the semen and seminal fluid can then be
stored at low temperature for use in artificial insemination.
[0013] The semen in this aspect of the invention may already have
been boosted in function or viability by virtue of the animal
having antioxidant and/or PUFA administered to it. The PUFA is
preferably administered to the animal in an amount of at least 10
mg/kg of body weight, most preferably 10-45 mg/kg.
[0014] The invention also provides a method of enhancing the
function and/or viability of sperm, the method comprising
controlling the PUFA content of the sperm, preferably the plasma
membrane of the sperm, although the control of PUFA content of the
seminal plasma can also be of benefit. The PUFA content of the
plasma membrane can be controlled eg by adding PUFA or antioxidant
to the sperm directly or administering the PUFA or the antioxidant
to the animal's diet.
[0015] The invention also provides a method of combatting sperm
dysfunction, comprising controlling the PUFA content of the sperm,
preferably the content of the sperm plasma membrane, eg by exposing
the sperm to a PUFA or an antioxidant.
[0016] The term "combat" as used herein refers to the prevention of
a condition (ie prophylactic use) as well as treatment of an
existing condition to ameliorate that condition or to delay or
prevent its further deterioration.
[0017] The PUFA can be added direct to the ejaculate, or can be
administered to an animal to enhance the function and/or viability
of sperm from that animal. In such a case, the PUFA is preferably
administered in quantities of at least 10-45 mg/kg body weight. The
PUFA can be provided in substantially pure form or in combination
with a pharmaceutical carrier or excipient, or in impure form. For
example, the PUFA may be provided in the form of fish oil, or can
be extracted from brain tissue by conventional methods. The PUFA
may be incorporated into the fatty acid pool of the sperm, or may
remain in the seminal fluid in order to exert its beneficial
effects.
[0018] The PUPA is preferably a C18-C24 fatty acid.
[0019] The viability can be enhanced by increased mobility, cold
resistance or related factors.
[0020] Two embodiments of the invention include:
[0021] (i) the maximisation of male fertility in vivo through the
dietary manipulation of the lipid composition and/or antioxidant
capacities of the fresh ejaculate.
[0022] (ii) the development of effective antioxidant/lipid
additives for semen diluents and effective carrier systems for
inclusion of the additives into the sperm membrane in order to
ensure sperm viability in vitro and in vivo and fertility
capacities after storage.
MATERIALS AND METHODS
[0023] The investigations involved both avian (cockerel) and
mammalian (bull) species.
[0024] (i) Avian dietary treatments.
[0025] (a) Supplementation with alpha-linolenic acid (18:3
n-3).
[0026] Two groups, each of 15 male broiler breeders from the same
genetic stock, were purchased from a commercial breeder supplier.
The males were 21 weeks of age at the beginning of the experiment
and 72 weeks old at the end. The males were housed in single cages
in a controlled environment with a photoperiod of 13 hours light:
11 hours dark. They were each fed 130 g per day of feed with 12.5%
crude protein and 11.5 MJ/kg of ME. The control diet was
supplemented with soyabean oil (6% w/w of feed) and the 18:3 (n-3)
enriched diet was produced by supplementation with linseed oil (6%
w/w of feed), see Table 1. Regular lipid analysis of feed was
undertaken to establish the lipid and fatty acid composition. The
males were trained for semen collection from 21 weeks of age and
were milked routinely twice weekly throughout the experimental
period and three times on the weeks 24, 40 and 54 chosen for
laboratory analysis. Lipid analysis was performed on 5 pooled semen
samples.
[0027] (b) Dietary supplementation with docosahexaenoic acid (22:6
n-3).
[0028] Two groups, each of 12 male broiler breeders from the same
genetic stock were used. The males were 11 weeks of age at the
beginning of the experiment with semen being collected at 24 weeks
and 38 weeks of age. Housing, treatment and control diet were as
per example 1. The 22:6 (n-3) enriched diet was produced by
supplementation with a 22:6 (n-3) enriched fish oil extract (3% w/w
of feed), see Table 1. Semen collection was as described in
(i)(a).
[0029] A second trial involving dietary supplementation with 22:6
(n-3) was subsequently undertaken. Details of the diet, housing and
general management of the cockerels were as for the first trial.
The 22:6 (n-3) was delivered by the inclusion in the diet of the
fish oil at a rate of 5% w/w within the feed. Semen collection was
performed as per (i)(a) above with investigations of chemical and
physiological parameters being undertaken on samples at 24, 40 and
58 weeks of age. In this experiment there was a further
experimental group in which 22:6(n-3) was accompanied by the
inclusion of 200 mg/kg of .alpha.-tocopherol in the diet.
[0030] (c) Supplementation with gamma-linolenic acid
(18:3.n-6).
[0031] Two groups, each of 20 male broiler breeders from the same
genetic stock were used. The males were 21 weeks of age at the
beginning of the experiment. Housing, treatment and control diet
were as per (i)(a) above. The 18:3(n-6) enriched diet was produced
by supplementation with evening primrose oil (5% w/w of feed)
containing 9% w/w of 18:3 (n-6), see Table 1. Semen was collected
five times a week at 40 weeks of age. Lipid analyses was performed
on 7 individual samples.
[0032] (ii) Bull dietary treatments
[0033] Two groups, each of three Holstein/Freisian bulls from
Scottish Livestock Services AI Centre, Perth, Scotland comprised
the main locus of experimentation; in addition, observations were
made on a Belgian Blue bull, a breed known for its inherently low
level of male fertility. Each bull was kept under standard (and
conventional) conditions appropriate to a leading AI centre. All
the bulls were fed twice daily 3 kg of a standard diet delivering
12.5 MJ/kg ME and 15% crude protein. Following appropriate
collection and sampling of the ejaculates, each bull was then
switched to a diet, based on the standard diet, but which for each
3 kg delivered 90 g of a fish oil containing 25% 22:6 (n-3). Thus
with the average bull weighing 800 kg, delivery of 22:6 (n-3) was
some 45 mg per kg body weight. The bulls were then sampled after an
8 week period on this diet. The major fatty acid within the diets
are shown in Table 1. Semen was collected by artificial vagina.
[0034] (iii) Spermatozoa evaluation. In the case of the cockerels
sperm quality measurements were made at 24, 39 and 54 weeks of age.
Pooled semen samples of 3 ejaculates (5 replicates per group) were
analysed in each case. In the case of the bulls, semen was
collected every 2 weeks over the complete period of the experiment.
Within 20 minutes of collection appropriate semen parameters were
measured that included ejaculate volume, sperm concentration,
acrosomal integrity and motility using microscopic and Cellsoft
Computer Assisted Analysis. Fertility in the cockerels was assessed
by insemination of laying hens with a fixed dose of semen
(70.times.10.sup.6 cells/ml). Eggs were collected for 2 weeks for
groups (i)(a) and (i)(b) and 3 weeks for (i)(c) and incubated for 7
days before candling to record the presence of any embryo. Weekly
fertilities were measured in groups (i)(a) and (i)(b) and daily for
(i)(c).
[0035] (iv) Preparation of semen for lipid extraction. Semen was
diluted with an equal volume of 0.85% (w/v) sodium chloride
solution and centrifuged at 700 g for 20 minutes at 4.degree. C.
The upper diluted plasma layer was transferred to a fresh tube, the
wash procedure was repeated with 1 ml of 0.85% (w/v) sodium
chloride and the final cell pellet was re-suspended in 2 ml of
0:85% (w/v) sodium chloride. In order to obtain sufficient material
for analysis from each individual cockerel, the successive samples
obtained during the 3 week collection period were combined.
[0036] (v) Lipid analysis. Total lipids were extracted from the
spermatozoa preparations following homogenisation in a suitable
excess of chloroform:methanol (2:1 v/v). The lipids were
fractionated into their major classes (phospholipid, free
cholesterol, triacylglycerol, free fatty acids and cholesterol
ester) by thin layer chromatography on silica gel G using a solvent
system of hexane:diethyl ether:formic acid (80:20:1 v/v/v).
Following visualisation under UV light after spraying with 0.1% w/v
solution of 2,7-dichlorofluorescein in methanol, the separated
bands were scraped from the plates. Phospholipid was eluted from
the silica by washing 3 times with 2 ml methanol and the other
lipid classes were similarly eluted with diethyl ether. The
esterified lipid fractions were subjected to transmethylation by
refluxing with methanol:toluene:sulphuri- c acid (20:10:1 v/v/v) in
the presence of a pentadecanoic acid standard. The resultant fatty
acid methyl esters were analysed by 1 .mu.m injection, via a CP9010
auto sampler (Chrompack, London, UK), on to a 30 m.times.0.25 mm
diameter, 0.25 .mu.m film thickness Carbowax capillary column
(Alltech UK Ltd., Carnforth) fitted to a Chrompack CP9001
instrument (Chrompack, London, UK). Integration of the peaks using
an `EZ-Chrom` Data Handling System (Speck Analytical, Alloa, UK)
enabled the derivation of the fatty acid composition (% w/w of
total fatty acids). The amount of each lipid class was calculated
by comparison of the total fatty acid peak areas to that of the
pentadecanoic fatty acid standard. Free cholesterol was determined
by standard calorimetric assay (Boehringer, Lewes, UK). Individual
phospholipid classes were separated by high performance thin layer
chromatography (HPTLC) using a solvent system of methyl acetate:
isopropanol:chloroform:methanol:0.25% (w/v in H.sub.2O) KC1
(25:25:25:10:9 v/v/v/v/v). After charring, quantification was
performed by densitometry using a Shimadzu CS-9001 PC dual
wavelength flying spot thin layer scanner (Shimadzu Corporation,
Japan).
[0037] (vi) Statistical Analysis. Students t-test was used for all
statistical comparison. Data included 5+4 replicates respectively
for the cockerels and bulls included 5 replicates per group at each
collection period for lipid analysis and 15 and 8 replicates per
group at each collection period for semen evaluation. For cockerels
in groups (i)(a) and (i)(b) 5 replicates within each week of egg
collection at each collection period were used for assessment of
fertility and for cockerels in group (i)(c) 7 replicates were used
for assessment of fertility.
[0038] (vii) Bull semen diluent
[0039] Bulls of known reproductive performance were selected from
Holstein/Fresian and Belgian Blue breeds. Both groups were known to
exhibit problems with routine freezing of their semen, particularly
with respect to post-freeze survival of spermatozoa and maintenance
of acrosomal integrity. The bulls were 5-6 years of age and
housed/fed according to accepted commercial AI practice.
[0040] .alpha.-tocopherol additives for addition to fresh diluted
ejaculates were as follows: treatment A, (control) no
.alpha.-tocopherol, no semen diluent; treatment B, 10 mg/ml
.alpha.-tocopherol, skimmed milk diluent; treatment C, 1 mg/ml
.alpha.-tocopherol, skimmed milk diluent; treatment D, 10 mg/ml
.alpha.-tocopherol, egg yolk/biosophus plus diluent; treatment E, 1
mg/ml .alpha.-tocopherol, egg yolk/biosophus plus diluent;
treatment F, 10 mg/ml .alpha.-tocopherol, egg yolk/0.85% (w/v)
saline diluent; treatment G, 1 mg/ml, egg yolk/0.85% (w/v) saline
diluent.
[0041] Additive preparation: (.alpha.-tocopherol in milk buffer. To
prepare the diluent, 5-50 mg of DL-.alpha.-tocopherol was carefully
weighed into a fresh test tube. Immediately afterwards 5 ml of
fresh skimmed milk buffer was added and the preparation mixed
thoroughly. To disperse and ensure complete solubilisation of the
vitamin in the milk the preparation was homogenised thoroughly for
30-40 seconds followed by 10-15 seconds of sonication until a
clean, milky texture was obtained. The contents of the test tube
were then carefully poured into a darkened glass vial, plugged and
stoppered. The vial was stored immediately at 4.degree. C. and out
of any direct sunlight to keep the vitamin and milk in the best
condition for addition to the semen.
[0042] .alpha.-tocopherol in egg yolk. Fresh egg yolk was used to
aid in solubilisation of the lipid-soluble .alpha.-tocopherol. A
stock solution of Biosophus Plus 1:4 (v/v) in distilled water was
prepared and mixed thoroughly by manual inversion. A few drops of
egg yolk were placed in the bottom of a clean test-tube and 5-50 mg
of .alpha.-tocopherol were carefully weighed with the drops being
placed directly onto the egg yolk. The resultant mixture was then
diluted with 5.0 ml of the Biosophus Plus solution and homogenised
and stored as described above.
[0043] .alpha.-tocopherol in saline buffer. This was prepared as
described above using 5.0 ml of physiological saline (0.85% sodium
chloride w/v).
[0044] In all cases the uniformity of distribution of the
.alpha.-tocopherol throughout the diluent was confirmed before use
by sub-sampling and appropriate analysis based on high performance
liquid chromatography.
[0045] Semen dilutions. Fresh semen from each bull was placed in a
water bath at 37.degree. C. and treated as per routine semen
preparation procedures according to commercial AI practice. Each
ejaculate was divided equally into the required aliquots for the
addition of the additives. 100 ul of each additive was added to 1
ml of fresh semen. Semen straws were prepared containing 200 ul of
semen plus diluent with a concentration of 2.5.times.10.sup.7
spermatozoa per straw. For each treatment 10 straws were prepared,
half being used for in vitro pre-freeze determinations and the
remainder stored at -196.degree. C. for post-freeze determinations
7 days later.
[0046] Results were obtained from 4 separate collection periods per
bull. Artificial insemination and assessments of in vivo fertility
procedures were performed according to standard AI practices. All
analytical procedures were undertaken as per standard
methodologies.
[0047] (viii) Avian semen diluent.
[0048] In the case of the avian a single carrier for the
.alpha.-tocopherol in the semen diluent was assessed. It was based
on the use of seminal plasma harvested from ejaculates of donor
birds of the same breed/stock on which the tests were to be made.
The seminal plasma was harvested by appropriate centrifugation of
fresh semen and in particular, extreme care was taken to ensure the
complete absence of any contaminating cells.
[0049] To 10 mg of .alpha.-tocopherol in an appropriate clean glass
tube was added 5-10 ml of the seminal plasma. The whole was then
homogenised for 3-5 minutes followed by sonication for 1-2 minutes
to ensure thorough mixing. From this stock solution, varying
amounts were added to diluted fresh semen to give a final
concentration of between 10-500 ug .alpha.-tocopherol per ml of
semen. The semen was then exposed to combinations of a selection of
storage conditions embracing temperatures of 4.degree. and
37.degree. C. and times of 6, 12, 24, 48 and 72 hours. Following
exposure the ejaculates were evaluated for in vivo fertility and in
vitro assessment e.g. live sperm numbers, motility, chemical
parameters by standard microscopic and analytical procedures but to
include also specific tests of sperm viability based on
measurements of membrane integrity by ethidium bromide and
respiration using tetrazolium (reductase activity). A further test
of sperm viability promotion was undertaken involving the
comparison of these measurements in the presence or absence of
Fe.sup.++ as a stimulus for oxidation.
[0050] Statistical Analysis
[0051] Students t-test was used for all statistical comparisons.
Analysis of variance and correlations were undertaken as
appropriate.
RESULTS
[0052] Dietary Supplementation
[0053] (i) Cockerel
[0054] (a) Supplementation with alpha-linolenic acid (18:3
n-3).
[0055] The effects of dietary supplementation with 18:3 (n-3) on
the characteristics of the semen samples obtained from cockerels at
24, 40 and 54 weeks of age are shown in Table 2. For the cockerels
on the control diet, the concentration of spermatozoa in the semen
increased considerably between 24 and 40 weeks of age and then
decreased markedly to 54 weeks. Dietary supplementation with 18:3
(n-3) significantly increased spermatozoa concentration at 54
weeks. Also, at 54 weeks the spermatozoa motility was significantly
increased by the supplementation. In the control cockerels,
fertility increased to a maximum at 40 weeks but had decreased by
54 weeks. The n-3 supplementation resulted in a significant
increase in week 1 fertility at 40 weeks. Although fertility was
not enhanced at 54 weeks, observations of the fertility at 72 weeks
(not shown) were enhanced by n-3 supplementation.
[0056] The proportions of the major lipid and phospholipid classes
of the spermatozoa are given in Table 3. The concentration of the
total lipid in the spermatozoan cells increased continually with
age; although not significant these values were higher for the
supplemented birds at 40 and 54 weeks. Phospholipid was the major
lipid class at all stages. However, the proportion of phospholipid
decreased considerably with age. Supplementation with 18:3 (n-3)
did not result in any dramatic effects on the proportions of the
major lipid classes. Phosphatidyl choline and phosphatidyl
ethanolamine were the main classes of phospholipid but there were
no major effects of n-3 supplementation on the proportions of the
major phospholipid classes.
[0057] The polyunsaturated fatty acid compositions of the total
spermatozoan phospholipid from control and n-3 supplemented
cockerels are presented in Table 4. The major polyunsaturated fatty
acids in the control samples were 20:4 (n-6) and 22:4 (n-6); the
phospholipids were almost devoid of n-3 polyunsaturates apart from
the presence of very low levels (approximately 2% w/w) of 22:6
(n-3). Dietary supplementation with 18:3 (n-3) resulted in small
but significant effects on these fatty acid profiles. Thus n-3
supplementation increased the levels of 22:5 (n-3) at 40 and 54
weeks and 22:6 (n-3) at 54 weeks. Whereas the levels of 22:6 (n-3)
within the phosphatidyl ethanolamine fraction, normally the major
carrier of the acid, in the control samples at weeks 40 and 54 were
negligible, within the treated birds the levels were 2.2 and 3.1%
respectively of total fatty acids present. However, most notably
supplementation resulted in considerable decreases in the C20-22
n-6:n-3 ratios at weeks 40 and 54.
[0058] (b) Supplementation with docosahexaenoic acid (22:6
n-3).
[0059] Supplementation of the cockerels with 22:6 (n-3) resulted in
an intensive change in overall appearance and visual parameters of
assessment of the ejaculates at 40 and 58 weeks of age. Sperm
concentration displayed a rise from 2.08.times.10.sup.9 /ml for the
control group to 2.23 and 2.40.times.10.sup.9/ml at 40 and 58 weeks
of age respectively for the treated group. A significant increase
in fertility as measured by AI was observed, 40.5.+-.6.6(SE),
55.4.+-.4.2 and 68.5.+-.4.9 respectively. As can be seen in Tables
5 and 6 the levels of 22:6 (n-3) within the total phospholipid of
the sperm and throughout all the major individual phospholipid
moieties underwent a significant increase to accompany this
increase in fertility. At the same time there were extensive and
appropriate reductions in total n-6:n-3 fatty acid ratios. At
slaughter at 60 weeks of age testis (single) weight in the control
group was 15.1 g.+-.1.4 compared with 22.3 g.+-.3.0 for the
cockerels supplemented with 22:6 (n-3) with no change in body
weights.
[0060] In the second experiment involving 22:6 (n-3)
supplementation, the compositional changes within the sperm were
similar in both absolute and relative terms to those described for
the first experiment (see Tables 5 and 6). Although the inclusion
of .alpha.-tocopherol did not enhance to any significant degree the
levels of polyunsaturates, the content of .alpha.-tocopherol in the
spermatozoa was significantly increased by 60-70% above the other
groups. Inherently the birds used in this experiment were more
fertile (increased sperm number per unit volume of ejaculate etc.)
than for the first experiments. Again supplementation of the
cockerels with 22:6 (n-3) resulted in extensive changes in overall
appearance and visual parameters of assessment of the ejaculates at
40 and 58 weeks of age. Spermatozoa concentration and other major
parameters of fertility are shown in Table 7. As can be seen, semen
volume, total spermatozoa number and fresh and stored fertilities
were all significantly enhanced; relative spermatozoa motilities
were increased by some 15% The inclusion of .alpha.-tocopherol had
an added effect on fertility after storage. Testis weights (weights
of 2 testes per bird) were again significantly increased by 22:6
(n-3) treatment without any accompanying differences in body
weight. Investigations on the distribution of 22:6 (n-3) within the
spermatozoan cell showed a preferential incorporation of the acid
into the mitochondria. An important feature arising from the
dietary enhancement with 22:6 (n-3) was an improved fertility of
eggs during the 2nd week after artificial insemination. In
approximate terms this equated with an extra 1.7 eggs becoming
available over the whole 2 week period of the fertility
investigation compared to the control treatments.
[0061] Supplementation with gamma linolenic acid (18:3 n-6)
[0062] Fatty acid compositions of the major lipid fractions were
unchanged as a result of supplementation with 18:3 n-6. Major
spermatozoa features associated with increased fertility were
significantly increased by 18:3 n-6 supplementation (see Table 8);
these embraced motility and fertilities over 1st, 2nd and 3rd weeks
after artificial insemination. FIG. 1 shows the fertility rate on a
daily basis following a single insemination of a fixed dose of
10.times.7 spermatozoa. As can be seen a positive difference in
fertilities was prominent over the 2nd week in particular but also
over the initial part of the 3rd week following insemination by the
18:3 (n-6) group compared to the control. This difference equated
to an extra 2 fertile eggs per hen over the 2nd week following
artificial insemination.
[0063] (ii) Bull
[0064] As can be seen from a comparison of the various parameters
of sperm evaluation on the 2 diets (see Table 9), the switch to the
diet to which had been added 22:6 (n-3) had a significant effect
across the board on sperm fertility characteristics. Appropriately
the levels of 22:6 (n-3) within the phosphatidyl ethanolamine
fraction, that is the major phospholipid moiety associated with
22:6 (n-3), underwent a significant increase from a pre treatment
level of 33.3.+-.1.0 (S.E.) to 60.6.+-.0.7 (S.E.) following
treatment (p<0.001). Due to commercial considerations,
appropriate fatty acid analysis on the ejaculate of the single
Belgian Blue bull was not possible.
[0065] Semen Diluents
[0066] (i) Bull
[0067] Determination of .alpha.-tocopherol concentrations within
the semen routinely showed that samples from group A (control)
displayed low levels only of .alpha.-tocopherol, the levels
increasing by some 100 fold with 1 mg/ml .alpha.-tocopherol
supplementation and 1000 fold with 10 mg/ml .alpha.-tocopherol
supplementation. Highest levels of malondialdehyde within the semen
following storage at -196.degree. C. were associated with group A
and lowest levels with 10 mg/ml .alpha.-tocopherol supplementation.
In vitro parameters of semen quality prior to freezing for the 2
groups of bulls are shown in Tables 10 and 11. Theprotocol for
commercial semen sale requires a minimum of 3.5 and 35% for
motility and PPM values respectively for both fresh and frozen
semen analysis. Semen failing to meet such requirements would be
discarded. As can be seen, semen from Group A (control) exhibited
values approximately equal to these minima. By comparison 3 of the
treatments showed a selection of improvements in motility and
greater survival characteristics. In vitro parameters of semen
quality post freezing at -196.degree. C. for the 2 groups of bulls
are shown in Tables 12 and 13. Although the quality of the Belgian
Blue semen was not acceptable for commercial use, improvements were
evident as a result of treatment. Marked improvements in parameters
were exhibited by the Holstein/Fresian bulls.
[0068] Table 14 gives the results from insemination using Group B
semen samples from the Belgian Blue bulls following storage at
-196.degree. C. As can be seen, in field experiments the treated
semen resulted in a considerable enhancement of pregnancy.
[0069] Cockerel
[0070] The inclusion of .alpha.-tocopherol into the diluent, using
harvested seminal plasma as a carrier, significantly increased
resistance of the spermatozoa lipids to oxidation as based on an
extensive range of biochemical parameters. Thus the level of 22:4,
the most susceptible fatty acid to the lipid oxidation, in the
spermatozoa phospholipids after storage was significantly higher
compared to the control spermatozoa without vitamin E
supplementation (see FIG. 2). The stabilising effect was seen
during the full 24-72 hours of the spermatozoa storage at 4.degree.
C. The increase in storage temperature caused a pronounced
reduction in the level of the long chain polyunsaturated fatty
acids in the phospholipids. Under such conditions the diluent was
also effective as a protective agent against oxidation (see FIG.
3).
[0071] Storage was also associated with reduction of reductase
activity, a feature that reflects damage to the respiratory chain
of mitochondria of the spermatozoa involving peroxidation of the
mitochondria lipids. The diluent clearly preserved the spermatozoa
mitochondria lipids from oxidation and promoted reductase activity
at both 4.degree. C. and 37.degree. C. (see FIGS. 4 and 5
respectively).
[0072] The main problem of spermatozoa storage is membrane damage
as a result of lipid peroxidation. Under such conditions membrane
permeability is dramatically increased and fertilisation capacity
reduced. As can be seen from FIGS. 6 and 7 the inclusion of the
diluent significantly increased sperm membrane integrity after
storage at both 4.degree. C. and 37.degree. C. The effect of all
these parameter changes under normal conditions is to diminish
considerably sperm motility after storage. A pronounced protective
effect of the diluent on spermatozoa motility following storage at
4.degree. C. and 37.degree. C. was observed (see FIGS. 8 and 9).
That vitamin E was distributed uniformly throughout the semen was
verified by appropriate determination of .alpha.-tocopherol from
different parts of diluted semen (see FIG. 10). It was significant
that after incubation of the spermatozoa, some 8% of the
.alpha.-tocopherol had become incorporated into the membranes (See
FIG. 11) and it was not possible to remove it during 3-5
consecutive washings with pure diluent. Confirmation of the
protective effect of the diluent against lipid peroxidation sperm
was further obtained by incubation of the spermatozoa in the
presence of Fe.sup.2++ at 37.degree. C. The results obtained (see
FIG. 12) indicate that malondialdehyde accumulation was less than
one third that of the control spermatozoa.
DISCUSSION
[0073] There is an overwhelming preponderance of linoleic (18:2
n-6) in proprietary feeds of domestic farm animals. Other fatty
acids of the n-6 series and those of the n-3 series are notable by
their virtual absence. That such a predominance of linoleic acid
may not always be wholly beneficial to the well-being and health of
the animal through effects upon tissue fatty acid composition and
aspects of metabolism is now being asked. With such a high-profile
presence of long chain polyunsaturated fatty acids of the n-3
series in mammalian sperm lipids, it is suggested that the
alteration of the current fatty acid profile of animal proprietary
feeds towards increasing levels of acids of the n-3 series may be
highly relevant to the ontogeny of the characteristic fatty acid
profiles and subsequent function of the sperm. Similarly, in the
avian the high profile presence of C20 and C22 polyunsaturates of
the n-6 series would also suggest the need to attempt their
improved availability. Presently reported are the results from
experiments designed to evaluate the deliberate enhancement of the
diets of the cockerel and bull with fatty acids of the n-3 series
and n-6 upon the lipid/fatty acid profile of the spermatozoa and
associated changes to parameters of spermatozoa function and
fertility.
[0074] It is clear from the analyses that initial lipid/fatty acid
compositions of the spermatozoa of the 2 species conformed to that
which has been previously reported. Thus, whereas in both species
the lipids of the spermatozoa displayed extremely high levels of
polyunsaturates, in the bull there was predominance of 22:6 (n-3)
and in the cockerel 22:4 (n-6). The apparent substitution of 22:4
(n-6) for 22:6 (n-3) in the cockerel can be suggested to be the
reaction to an almost complete domination in the diet of linoeic
acid (18:2, n-6) and thereby determining that 22:4 (n-6) as opposed
to 22:6 (n-3) be the long chain polyunsaturate for spermatozoa
inclusion.
[0075] The inclusion of the n-3 fatty acids in the diet was to
increase significantly their levels within the spermatozoa and to
have extensive beneficial effects on parameters of spermatozoa
function and therefore male fertility in the species. Although in
the case of the cockerel these was a marked difference in the
levels attainable within the spermatozoa of the long chain n-3
polyunsaturates, nevertheless effects on spermatozoa parameters
were very positive. 22:6 (n-3) is an extensively available fatty
acid. It is clear from the present results that deliberate
enhancement of this acid within the diet of the cockerel and bull
and also long chain polyunsaturates of the n-6 series in the
cockerel presents a simple and effective means of promoting a range
of parameters that lead to increased spermatozoa quality, output
and viability at ejaculation. In the case of the cockerel, the
result was to lead to a significant increase in output of the
fertile eggs from the hen, a most important feature to commercial
production. Similarly in the case of the bull, a dramatic decrease
in "non return" rates of heifers was observed.
[0076] Intensive animal production systems require an efficient
insemination service, both natural and artificial. This is clearly
dependent not only upon maximising the initial fertility of fresh
ejaculates but also its maintenance during storage. The need exists
to extend the life of semen for a fresh delivery service and
enhance the ability to maintain spermatozoa function during the
following cryoscopic storage in all farm animal species.
[0077] The present data have clearly demonstrated the ability to
promote the maintenance of spermatozoa viability and function
following cryoscopic storage through the addition of
.alpha.-tocopherol, in particular through a unique carrier medium.
A very broad range of spermatozoa characteristics were able to be
increased compared with spermatozoa maintained under standard
cryoscopic AI conditions. Apart from measurements in vivo, the
effect of the carrier/.alpha.-tocopherol medium was to prevent the
significant reductions that arise as a result of storage in a range
of biochemical and physiological features that are known to be
intimately associated with spermatozoa viability and function. The
data clearly demonstrates a means whereby a significant enhancement
of male fertility can be obtained following sperm storage in the
liquid state.
[0078] The present work therefore underlines 2 major vectors
through which male fertility in mammalian and avian species may be
significantly enhanced with appropriate and significant benefits to
subsequent stock production:
[0079] (i) by the deliberate manipulation of the spectrum and level
of long chain fatty acid combinations within the spermatozoa by
appropriate-dietary means.
[0080] (ii) by the addition to the ejaculate prior to cryoscopic
and hypothermic storage of .alpha.-tocopherol through a unique
carrier medium, including harvested seminal fluid lipids from donor
animals.
[0081] It is clear that the invention is transferable across
species to include the human.
1TABLE 1 The major fatty acids (percent by weight of total) in the
diets. Cockerel Bull Diet 1 Diet 4 Diet 1 (soya- Diet 2 Diet 3
(evening (soya- Diet 2 major bean (linseed (fish primrose bean
(fish fatty acids: oil) oil) oil) oil) oil) oil) palmitic 12 10 22
10 15 20 (16:0) stearic (18:0) 4 4 6 2 3 4 oleic (18:1, 23 21 19 13
17 16 n-9) linoleic 50 30 20 62 52 32 (18:2, n-6) linlenic 6 34 3 2
6 4 (18:3, n-3) docosa- <1 <1 14 -- <1 11 hexaenoic (22:6,
n-3) 5 (18:3 n-6)
[0082]
2TABLE 2 The effect of linolenic acid (18:3, n-3) supplementation
on semen characteristics of the cockerel. Week 24 Week 40 Week 54
Control Treated Control Treated Control Treated Sperm conc. 4.7
.+-. 5.0 .+-. 7.7 .+-. 7.6 .+-. 5.1 .+-. 6.8 .+-. (10.sup.9
cells/ml) 0.5 0.6 0.6 0.6 0.9 0.7* motility (1%) 56.4 .+-. 56.5
.+-. 54.5 .+-. 62.5 .+-. 33.8 .+-. 53.9 .+-. 4.1 4.4 3.8 5.1 3.9
4.7** fertility (%) Week 1 68.3 .+-. 62.7 .+-. 82.8 .+-. 96.8 .+-.
74.4 .+-. 76.8 .+-. 4.9 9.1 4.9 3.2* 4.6 3.8 Week 2 45.0 .+-. 58.4
.+-. 61.7 .+-. 57.5 .+-. 47.9 .+-. 54.6 .+-. 7.1 10.8 4.7 7.3 6.4
8.4 Values are means .+-. standard error. Significance of
difference between control and treated: *p < 0.05, **p <
0.01
[0083]
3TABLE 3 The effect of linolenic acid (18:3 n-3) supplementation on
the concentration and proportion of the major lipid and
phospholipid classes (percent by weight of total) in the
spermatozoa of the cockerel. Week 24 Week 40 Week 54 Control
Treated Control Treated Control Treated Total lipid 261.2 .+-.
240.0 .+-. 274.4 .+-. 316.0 .+-. 364.4 .+-. 427.7 .+-. .mu.g/109
cells 12.1 27.6 16.9 44.0 75.3 84.1 Lipid class (% w/w of total
lipid) PL 60.1 .+-. 68.3 .+-. 69.2 .+-. 68.5 .+-. 57.7 .+-. 57.4
.+-. 2.3 4.4 2.1 1.6 1.4 3.4 FC 12.6 .+-. 12.1 .+-. 12.9 .+-. 17.8
.+-. 24.8 .+-. 23.8 .+-. 1.4 2.6 0.6 0.9 1.8 1.8 FFA 4.9 .+-. 5.9
.+-. 6.9 .+-. 5.3 .+-. 9.0 .+-. 4.9 .+-. 1.6 1.0 1.2 0.5 1.8 1.4 TG
9.3 .+-. 5.3 .+-. 3.8 .+-. 3.2 .+-. 3.1 .+-. 4.7 .+-. 3.1 2.8 1.1
2.1 0.7 1.3 CE 13.1 .+-. 8.4 .+-. 7.2 .+-. 5.2 .+-. 8.5 .+-. 9.2
.+-. 3.6 3.9 1.2 0.8 0.6 2.2 Phospholipid class (% w/w of total
phospholipid) PC 32.3 .+-. 33.4 .+-. 25.4 .+-. 26.6 .+-. 34.7 .+-.
32.4 .+-. 1.5 1.1 1.6 1.3 1.2 1.1 PE 33.1 .+-. 31.7 .+-. 33.8 .+-.
32.9 .+-. 31.1 .+-. 32.9 .+-. 1.6 1.2 1.2 0.8 0.4 0.9 PS 19.2 .+-.
18.9 .+-. 24.4 .+-. 22.3 .+-. 20.9 .+-. 21.6 .+-. 2.1 0.7 0.7 0.8
0.8 0.5 Sph 10.5 .+-. 11.9 .+-. 11.5 .+-. 12.9 .+-. 8.4 .+-. 9.4
.+-. 1.1 1.3 0.4 2.9 0.5 0.5 CL 4.9 .+-. 4.1 .+-. 4.8 .+-. 5.2 .+-.
4.9 .+-. 3.7 .+-. 0.5 0.5 0.4 0.8 0.3 0.2 Values are means .+-.
standard error. PL = phospholipid; FC = free cholesterol; FFA =
free fatty acid; TG = triacylglycerol; CE = cholesterol ester PC =
phosphatidyl choline; PE = phosphatidyl ethanolamine; PS =
phosphatidyl serine; Sph = sphingomyelin; CL = cardiolipin.
[0084]
4TABLE 4 The effect of linolenic acid (18:3, n-3) supplementation
on the polyunsaturated fatty acid concentrations (percent by weight
of total fatty acids) within the phospholipid fraction of the
spermatozoa of the cockerel. Week 24 Week 40 Week 54 Control
Treated Control Treated Control Treated n-6 acids: 18:2 (n-6) 2.7
.+-. 2.4 .+-. 3.4 .+-. 2.3 .+-. 4.7 .+-. 3.7 .+-. 0.2 0.1 0.5 0.1
0.4 0.3 20:4 (n-6) 12.5 .+-. 13.1 .+-. 11.7 .+-. 12.1 .+-. 11.9
.+-. 11.7 .+-. 0.4 0.4 0.4 0.5 0.2 0.2 22:4 (n-6) 22.8 .+-. 23.0
.+-. 22.9 .+-. 19.9 .+-. 21.7 .+-. 19.2 .+-. 1.1 0.8 1.0 0.8 1.4
0.6 n-3 acids: 18:3 (n-3) 0.8 .+-. 1.2 .+-. nd 0.4 .+-. nd nd 0.5
0.7 0.03 22:5 (n-3) 1.0 .+-. 0.8 .+-. 1.0 .+-. 5.3 .+-. 0.8 .+-.
3.4 .+-. 0.2 0.2 0.1 0.9** 0.05 0.1** 22:6 (n-3) 2.2 .+-. 2.4 .+-.
2.5 .+-. 2.3 .+-. 1.9 .+-. 2.4 .+-. 0.1 0.1 0.2 0.1 0.1 0.1* C20-22
n6/n-3 10.5 .+-. 10.0 .+-. 10.9 .+-. 4.5 .+-. 14.5 .+-. 5.6 .+-.
ratio 1.4 1.8 0.5 0.6** 0.6 0.2** Values are means .+-. standard
error; nd = not detectable. Significance of difference between
control and treated: *p < 0.05; **p < 0.01.
[0085]
5TABLE 5 The effect of docosahexaenoic acid (22:6 n-3)
supplementation on the C20 and C22 polyunsaturated fatty acid
concentrations (percent by weight of total fatty acids) within the
phospholipid fraction of the spermatozoa of the cockerel. Week 24
Week 40 Week 58 Control Treated Control Treated Control Treated
20:4 (n-6) 13.0 .+-. 8.9 .+-. 11.4 .+-. 8.6 .+-. 10.7 .+-. 8.3 .+-.
0.1 0.1*** 0.3 0.2*** 0.2 0.2*** 22:4 (n-6) 19.5 .+-. 8.5 .+-. 21.7
.+-. 15.0 .+-. 18.2 .+-. 12.4 .+-. 0.8 0.4*** 0.2 0.6*** 0.4 0.5***
22:5 (n-3) nil nil nil nil 1.9 .+-. 3.1 .+-. 0.01 0.1*** 22:6 (n-3)
4.7 .+-. 13.3 .+-. 3.8 .+-. 10.1 .+-. 5.1 .+-. 9.1 .+-. 0.1 0.5***
1.1 0.2*** 0.2 0.3*** C20-22 n-6/n-3 6.9 1.3*** 8.7 2.3** 4.1 1.7**
ratio Values are means .+-. standard error. nd = not detectable.
Significance of difference between control and treated: ***p <
0.001.
[0086]
6TABLE 6 The effects of docosahexaenoic acid (22:6 n-3)
supplementation on the concentration of 22:6 (n-3) (percent by
weight of total fatty acid) within the major phospholipid fractions
of the spermatozoa of the cockerel. Week 24 Week 40 Week 58 C T C T
C T PC 1.7 .+-. 0.3 7.0 .+-. 1.1 .+-. 0.3 5.2 .+-. 2.6 .+-. 0.2 5.7
.+-. 0.3*** 0.4*** 2.7 PE 6.7 .+-. 0.7 22.6 .+-. 6.1 .+-. 1.2 16.4
.+-. 9.4 .+-. 0.2 14.9 .+-. 1.4*** 1.0*** 1.1** PS 6.1 .+-. 0.9
20.5 .+-. 5.6 .+-. 0.4 17.5 .+-. 7.1 .+-. 0.7 13.1 .+-. 2.0***
1.1*** 0.7*** Sp1 4.8 .+-. 2.6 16.4 .+-. 25.5 .+-. 3.6 11.2 .+-.
14.4 .+-. 2.2 17.7 .+-. 2.3* 3.6* 2.3 CL 9.2 .+-. 0.2 24.2 .+-. 5.3
.+-. 0.8 14.0 .+-. 2.7 .+-. 0.3 14.3 .+-. 0.7*** 1.8** 1.6** Values
are means .+-. standard error. Significance of difference between
control and treated: **p < 0.01; ***p < 0.001. PL =
phospholipid; FC = free cholesterol; FFA = free fatty acid; TG =
triacylglycerol; CE = cholesterol ester PC = phosphatidyl choline;
PE = phosphatidyl ethanolamine; PS = phosphatidyl serine; Sph =
sphingomyelin; CL = cardiolipin.
[0087]
7TABLE 7 The effect of docosahexaenoic acid (22:6 n-3)
supplementation on the major cockerel's sperm parameters at 50
weeks of age. The results of the 2nd experiment. Control Diet
(maize oil) DHA DHA + Vit. E Semen volume, ml 0.25 0.45** 0.40**
Spermatozoa concentration 3.05 3.11 3.01 10.sup.9/ml Total number
of 0.763 1.400** 1.204** spermatozoa, 10.sup.9/ejaculate
Fertilizing capacity of the fresh 80.6 84.8* 86.9** semen, %
Fertilizing capacity of stored 70.4 69.6 77.9** 24 h at 4.degree.
C. semen % Testes weight, g 20.95 30.11** 37.14** Body weight, kg
5.37 5.69 5.89 Values are means. Significance of differences
between control and treated groups */-P < 0.05; **/-P, 0.01
[0088]
8TABLE 8 The effect of GLA (18:3 n-6) supplementation on the major
cockerel's sperm parameters at 40 weeks of age. Sperm parameters
Control GLA volume ml 0.62 .+-. 0.04 0.t6 .+-. 0.07 concentration
10.sup.9/ml 3.70 .+-. 0.20 2.74 .+-. 0.37 total sperm
10.sup.9/ejaculate 2.28 .+-. 0.19 1.84 .+-. 0.35 motility % 48.9
.+-. 3.11 54.0 .+-. 3.70 fertility 1st week.sup.2 92.8 .+-. 2.58
90.3 .+-. 2.15 fertility 2nd week.sup.3 58.1 .+-. 5.46 74.9 .+-.
5.18 fertility 3rd week.sup.4 13.9 .+-. 3.32 18.9 .+-. 5.18
.sup.2fertility of the 1st week after Al, .sup.3fertility of the
2nd week after Al, .sup.4fertility of the 3rd week after Al.
[0089]
9TABLE 9 The effect of docosahexaenoic acid (22:6, n-3)
supplementation on semen characteristics of the bull. Sperm conc.
Standard Standard Acrosomal (10.sup.9 cells/ml) Motility (%) PPM
(%) integrity (%) Fresian/ Holstein: pre-treatment 0.6 .+-. 0.1 3.3
.+-. 0.2 21.7 .+-. 4.4 70.0 .+-. 1.2 post- 0.9 .+-. 0.1* 4.0 .+-.
0.1* 36.0 .+-. 0.6* 90.0 .+-. 2.0*** treatment Belgian Blue:
pre-treatment 1.27 3.4 25 68 post- 2.82 4.0 36 80 treatment Values
are mean .+-. standard error. Significance of difference between
pre and post-treatment: *p < 0.05; **p < 0.01.
[0090]
10TABLE 10 Fresh semen in vitro characteristics of the Belgian
Blue. Citrate test Standard drop treatment motility PPM motility
PPM A 3 28 4 39 B 4* 38* 4 40* C 3.5 35* 4 38 D 3 30* 4 37 E 3 27 4
35 F 3 15 3.5 34 G 3 18 3.5 36 *Parameters greater than those of
the control
[0091]
11TABLE 11 Fresh semen in vitro characteristics of the
Holstein/Fresian. Citrate test Standard drop treatment motility PPM
motility PPM A 3.5 36 3.5 35 B 4* 37* 4* 38* C 4* 35 4* 36* D 3 36
3.5 36* E 3.5 35 3.5 34 F 4 38* 3.5 35 G 3.5 36 4* 37* *Parameters
greater than those of the control
[0092]
12TABLE 12 Frozen semen in vitro characteristics of the Belgian
Blue following storage at -196.degree. C. treat- Citrate test
Standard drop Acresomal integrity % ment motility PPM motility PPM
abnormal non intact intact A 3 22 3 10 31 25 72 B 3 20 2.5 13* 37
29 71 C 3 15 2.5 13* 33 29 71 D 3.5 15 2 7 30 28 72 E 2 17 2.5 12*
37 39 61 F 2.5 20 3 8 14* 34 56 G 2 25* 3 10 14* 56 44 *Parameters
greater than those of the control.
[0093]
13TABLE 13 Frozen semen in vitro characteristics of the
Holstein/Fresian following storage at -196.degree. C. treat-
Citrate test Standard drop Acresomal integrity % ment motility PPM
motility PPM abnormal non intact intact A 3.5 34 3 23 16 18 82 B 4*
35* 3.5* 29* 13 14* 86* C 3.5 30 3.5* 22 14* 17* 83* 0 3.5 30 3 18
10* 18 82 E 3 25 3 20 16 36 64 F 3 10 2.5 15 1 0* 28 72 G 3.5 31 3
14 13* 18 82 *Paramerers greater than those of the control.
[0094]
14TABLE 14 in vivo inseminations performed an synchronised heifers.
Treatment A B Trial 1: % heifers pregnant 56 64 Trial 2: % heifers
pregnant 31 55
* * * * *